Methods for diagnosing CNS disorders with fusion antibodies that cross the blood-brain barrier in both directions

ABSTRACT

The invention provides diagnostic and therapeutic macromolecular compositions that cross the blood-brain barrier, in some embodiments in both directions, while allowing their activity to remain substantially intact once across the barrier. Also provided are methods for using such compositions in the diagnosis or treatment of CNS disorders such as Alzheimer&#39;s disease.

RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.11/841,623, entitled “Fusion Antibodies that Cross the Blood-BrainBarrier in Both Directions,” filed Aug. 20, 2007, now U.S. Pat. No.7,741,446, which claims the benefit of U.S. Provisional Application No.60/822,825, entitled “Agents for Blood-Brain Barrier Delivery,” filedAug. 18, 2006, the contents of both of which are incorporated byreference in their entirety.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with the support of the United States governmentunder Grant number R43-NS-051857 by the National Institutes of Health.The United States Government may have certain rights in the invention.

REFERENCE TO A SEQUENCE LISTING SUBMITTED ELECTRONICALLY ANDINCORPORATION BY REFERENCE OF THE CONTENTS OF THE ELECTRONIC SUBMISSION

An electronic submission, containing the Sequence Listing for SEQ IDNO:1 through SEQ ID NO: 69 disclosed herein, and in computer readableform, as well as a paper copy of the Sequence Listing, are submittedherewith, are hereby referred to herein, and are hereby incorporated byreference herein in their entireties, including the contents thereof.

BACKGROUND OF THE INVENTION

The blood-brain barrier (BBB), a protective endothelial tissuesurrounding the CNS, is a major impediment to the systemic delivery ofhigh molecular weight therapeutic and diagnostic agents (e.g.,antibodies) to the CNS. The present invention represents an advance inmacromolecular therapeutic compositions capable of crossing the BBB intothe CNS and in the use of such compositions in diagnostic andtherapeutic applications (e.g., treatment of Alzheimer's disease).

SUMMARY OF THE INVENTION

In one aspect provided herein is a method of diagnosis of a braindisorder comprising detecting a signal emitted by a composition in theCNS of an individual, wherein the composition comprises an antibody thatis capable of crossing the BBB from the blood to the brain andinteracting with a pathological substance associated with a braindisorder.

In some embodiments, the pathological substance is a protein. In someembodiments, the protein is Aβ amyloid, α-synuclein, huntingtin Protein,PrP prion protein, West Nile envelope protein, tumor necrosis factor(TNF) related apoptosis inducing ligand (TRAIL), Nogo A, HER2, epidermalgrowth factor receptor (EGFR), hepatocyte growth factor (HGF), droligodendrocyte surface antigen. In some embodiments, the protein is Aβamyloid.

In some embodiments, the above-described composition is labeled with asubstance that emits the signal. In some embodiments, the substance thatemits the signal is selected from the group consisting of positronemitters, radionuclide, and magnetic substances. In some embodiments thecomposition comprises a ScFv.

In some embodiments, the above-described method also includessystemically administering the composition to the individual prior tothe detecting step.

In another aspect provided herein is a composition of molecular weightgreater than about 1000 Daltons that is capable of crossing the BBB fromthe blood to the brain; and crossing the BBB from the brain to theblood.

In some embodiments the just-mentioned composition is also capable ofinteracting with a pathological substance associated with a braindisorder. In some embodiments, the pathological substance is of a typeselected from the group consisting of proteins, nucleic acids,carbohydrates, carbohydrate polymers, lipids, glycolipids, smallmolecules, and combinations thereof. In some embodiments, thepathological substance is a protein. In some embodiments, the protein isAβ amyloid, a synuclein, huntingtin Protein, PrP prion protein, WestNile envelope protein, tumor necrosis factor (TNF) related apoptosisinducing ligand (TRAIL), Nogo A, HER2, epidermal growth factor receptor(EGFR), hepatocyte growth factor (HGF), or oligodendrocyte surfaceantigen. In some embodiments, the protein is Aβ amyloid.

In some embodiments, the above-mentioned composition comprises a ScFv.In some embodiments, the third portion of the just-mentioned compositioncomprises a ScFv. In some embodiments, the first and second portions ofthe composition are part of an antibody structure.

In some embodiments, the brain disorder associated with theabove-mentioned pathological substance is Alzheimer's disease,Parkinson's disease, Huntington's disease, bovine spongiformencephalopathy, West Nile virus encephalitis, Neuro-AIDS, brain injury,spinal cord injury, metastatic cancer of the brain, metastatic breastcancer of the brain, primary cancer of the brain, or multiple sclerosis.

In yet another aspect provided herein is a method of treatingAlzheimer's disease comprising administering to a subject in needthereof a composition comprising a first portion that crosses the BBBfrom the blood to the brain via a first receptor-mediated BBB; a secondportion that crosses the BBB from the brain to the blood via a secondreceptor-mediated BBB transport system; and a third portion thatinteracts with Aβ peptide.

In some embodiments, the third portion of the above-mentionedcomposition comprises a ScFv. In some embodiments the first and secondportions of the composition are part of an antibody structure.

In some embodiments, the first receptor mediated transport system in theabove-described method is an insulin receptor, transferrin receptor,leptin receptor, lipoprotein receptor, or an IGF receptor.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1A. Diagram showing genetic engineering of a prokaryotic expressionvector encoding a single chain Fv (ScFv) antibody against the Aβ peptideof AD. The ScFv is derived from a murine (m) antibody against Aβ, and isdesignated the mAβScFv. The ScFv is comprised of a variable region ofthe heavy chain (VH) and a variable region of the light chain (VL)originating from the murine MAb against the Aβ peptide. The VH and VLare joined by a linker to form the ScFv. The gene encoding the VH cDNAis produced by the polymerase chain reaction (PCR) from RNA isolatedfrom the murine hybridoma secreting the anti-Aβ antibody, usingoligodeoxynucleotide (ODN) primers that are specific for the mouse VHisotype; following PCR, the VH gene is ligated into the pPC plasmid withT4 ligase. The gene encoding the VL cDNA is produced by PCR from RNAisolated from the murine hybridoma secreting the anti-Aβ antibody, usingODN primers that are specific for the mouse VL isotype; following PCR,the VL gene is ligated into the pPC plasmid with T4 ligase.

FIG. 1B. Diagram showing genetic engineering of a eukaryotic expressionplasmid encoding mAβScFv cDNA with an IgG signal peptide and Kozak site.The eukaryotic expression plasmid contains the cytomegalovirus (CMV)promoter and a polyA (pA) transcription termination sequence.

FIG. 2. Diagram showing the genetic engineering of a eukaryoticexpression plasmid encoding the mAβScFv cDNA that is fused at its 5′-endto the 3′-end of the cDNA encoding the heavy chain (HC) of the chimericHIRMAb (HC-1). Amp=ampicillin resistance gene; G418=neomycin resistancegene; ori=SV40 origin of replication; CMV=cytomegalovirus promoter;pA=poly A transcription termination sequence.

FIG. 3. Ethidium bromide staining agarose gels showing the size ofvarious constructs and PCR amplified cDNAs that are intermediates in theconstruction of the fusion gene encoding the fusion antibody where themAβScFv is fused at its amino terminus to the carboxyl terminus of theheavy chain of the chimeric HIRMAb. (A) Lanes 1 and 2, PCR amplified 0.4kb mAβScFv VH cDNA and PCR amplified 0.4 kb mAβScFv VL cDNA,respectively. PolyA+ RNA was isolated form the murine hybridomaexpressing the anti-Aβ MAb, subjected to reverse transcription and PCRamplification with VH and VL specific ODN primers described in Table 2.Lane 3, molecular weight (MW) size standards ranging from 1.4-0.1 kb.Lane 4, MW size standards ranging from 23-2.0 kb. (B) Lane 1, NcoI andHindIII digestion of pPC-mAβ-VH showing the expected band size of ˜0.4kb corresponding to the VH of the anti-Aβ MAb, and the backbone vector(˜3.0 kb). Lane 2, NcoI and HindIII digestion of pAP-xScFv cloningvector showing the expected band size of ˜3.5 kb corresponding to thevector backbone, and the xVH (˜0.4 kb), where xVH is the VH of anon-related ScFv contained in the original pAP-xScFv plasmid (FIG. 1A).Lanes 3 and 4 are same MW size standards as shown in panel A. (C) Lane1, MluI and NotI digestion of pPC-mAβ-VL showing the expected band sizeof ˜0.4 kb corresponding to the VL of the anti-Aβ MAb, and the backbonevector (˜3.0 kb). Lane 2, MluI and NotI digestion of p-mAβ-VH showingthe expected band size of ˜3.5 kb corresponding to the plasmid backbone,and xVL (˜0.4 kb), where xVL is the VL of a non-related ScFv containedin the original pAP-xScFv plasmid. Lanes 3 and 4 are same MW sizestandards as shown in panel A. (D) Lane 1, XhoI-EcoRI digestion of aeukaryotic expression plasmid, pCD, showing the expected band size of˜5.4 kb corresponding the linear backbone vector (total of 2 lanes).Lane 2, XhoI-EcoRI digestion of PCR generated mAβScFv cDNA showing theexpected band size of ˜0.8 kb (total of 3 lanes) and minor bands oflower and higher MW size. Lanes 3 and 4 are same MW size standards asshown in panel A. (E) Lane 1, PCR product showing the expected singleband of ˜0.8 kb corresponding to the mAβScFv cDNA to be used in theengineering of the HIRMAb-mAβScFv fusion antibody tandem expressionvector. Lanes 2 and 3 are same MW size standards as shown in panel A.(F) HindIII restriction endonuclease mapping of the HIRMAb-mAβScFvfusion antibody tandem vector showing the expected bands of 6.5, 3.6,0.5 and 0.4 kb, respectively. Lanes 2 and 3 are same MW size standardsas shown in panel A.

FIG. 4. Nucleotide sequence (SEQ ID NO: 11) of the VH of the murineanti-Aβ MAb derived by PCR from the murine hybridoma secreting theanti-Aβ MAb. The sequence was determined by DNA sequencing of thepPC-mAβ-VH plasmid (FIG. 1A).

FIG. 5. Amino acid sequence (SEQ ID NO: 12) of the VH of the murineanti-Aβ MAb derived by PCR from the murine hybridoma secreting theanti-Aβ MAb. The amino acid sequence is deduced from the nucleotidesequence (SEQ ID NO: 11). The amino acids underlined matched with theamino acid sequence of tryptic peptides obtained from the heavy chain ofthe anti-Aβ MAb isolated from the murine hybridoma. The amino acidsequences of CDR1, CDR2, and CDR3 of the anti-Aβ MAb heavy chain VH arein bold font.

FIG. 6. Nucleotide sequence (SEQ ID NO: 13) of the VL of the murineanti-Aβ MAb derived by PCR from the murine hybridoma secreting theanti-Aβ MAb. The sequence was determined by DNA sequencing of thepPC-mAβ-VL plasmid (FIG. 1A).

FIG. 7. Amino acid sequence (SEQ ID NO: 14) of the VH of the murineanti-Aβ MAb derived by PCR from the murine hybridoma secreting theanti-Aβ MAb. The amino acid sequence is deduced from the nucleotidesequence (SEQ ID NO: 13). The 11 amino acids at the amino terminus ofthe deduced sequence matched the amino acids observed with direct aminoacid sequencing of the light chain of the hybridoma generated murineanti-Aβ MAb, except a valine (V) residue was observed at position 2 ofthe light chain of the anti-Aβ MAb derived from the mouse hybridoma. Theamino acid sequences of CDR1, CDR2, and CDR3 of the anti-Aβ MAb lightchain VL are in bold font.

FIG. 8. Nucleotide sequence (SEQ ID NO: 15) of the anti-Aβ ScFv cDNAencoded by the prokaryotic expression plasmid. The sequence wasdetermined by DNA sequencing of the pPC-mAβScFv plasmid (FIG. 1A).

FIG. 9. Amino acid sequence (SEQ ID NO: 16) of the anti-Aβ ScFv cDNAencoded by the prokaryotic expression plasmid, and includes apoly-histidine (H) tail, and the 9E10 epitope (EQKLISEEDL) (SEQ IDNO:66) at the carboxyl terminus. The sequence was determined by DNAsequencing of the pPC-mAβScFv plasmid (FIG. 1A). The amino acid sequenceis deduced from the nucleotide sequence (SEQ ID NO: 15). The 17-aminoacid linker separating the VH and VL is underlined.

FIG. 10. Nucleotide sequence (SEQ ID NO: 17) of the anti-Aβ ScFv cDNAencoded by the eukaryotic expression plasmid. The sequence wasdetermined by DNA sequencing of the pCD-mAβScFv plasmid (FIG. 1B).

FIG. 11. Amino acid sequence (SEQ ID NO: 18) of the anti-Aβ ScFv cDNAencoded by the eukaryotic expression plasmid. The poly-histidine (H)tail at the carboxyl terminus has been removed. The mAβScFv is nowdownstream of a 19-amino acid IgG signal peptide, and the signal peptidesequence is underlined. The amino acid sequence is deduced from thenucleotide sequence (SEQ ID NO: 17).

FIG. 12. Nucleotide sequence (SEQ ID NO: 19) of the fusion genecomprised of the anti-Aβ ScFv cDNA fused to the 3′-end of the cDNAencoding the chimeric HIRMAb heavy chain encoded by the eukaryoticexpression plasmid. The sequence was determined by DNA sequencing of thepCD-HC-mAβScFv plasmid (FIG. 2).

FIG. 13. Amino acid sequence (SEQ ID NO: 20) of the HC-mAβScFv fusionprotein, where the anti-Aβ ScFv is fused to the carboxyl terminus of thechimeric HIRMAb heavy chain (HC) via a S-S linker, and S=serine. TheHC-mAβScFv fusion protein is downstream of a 19-amino acid IgG signalpeptide, and the signal peptide sequence is underlined. The amino acidsequence is deduced from the nucleotide sequence (SEQ ID NO: 19).

FIG. 14. Nucleotide sequence (SEQ ID NO: 21) of the site-directedmutagenized fusion gene comprised of the anti-Aβ ScFv cDNA fused to the3′-end of the cDNA encoding the chimeric HIRMAb heavy chain encoded bythe eukaryotic expression plasmid, pCD-HC-mAβScFv plasmid (FIG. 2). The‘A’ nucleotide has been mutagenized to a ‘G’ nucleotide at position1789; the site is underlined in the figure. This change in nucleotidesequence results in the I2V amino acid change in the first frameworkregion of the VL of the mAbScFv, as shown in FIG. 15.

FIG. 15. Amino acid sequence (SEQ ID NO: 22) of the site-directedmutagenized HC-mAβScFv fusion protein. PCR amplification of the VL ofthe mAβScFv produced a cDNA, which encoded isoleucine (I) at the 2position of the first framework region of the VL; the mutagenizednucleotide is underlined in the figure. However, direct amino acidsequence analysis of the amino terminus of the murine anti-Aβ MAb showeda valine (V) at this position. The isoleucine residue at this site waschanged to a valine by site-directed mutagenesis, and this change iscalled I2V. The amino acid sequence is deduced from the nucleotidesequence (SEQ ID NO: 21).

FIG. 16. Nucleotide sequence (SEQ ID NO: 23) of the site-directedmutagenized fusion gene comprised of the anti-Aβ ScFv cDNA fused to the3′-end of the cDNA encoding the chimeric HIRMAb heavy chain encoded bythe eukaryotic expression plasmid, pCD-HC-mAβScFv plasmid (FIG. 2). The‘AA’ nucleotides have been mutagenized to ‘GC’ nucleotides at positions1546-1547; the site is underlined in the figure. This change innucleotide sequence results in the N497A amino acid change in the secondcomplementarity determining region (CDR) of the VH of the mAbScFv, asshown in FIG. 17.

FIG. 17. Amino acid sequence (SEQ ID NO: 24) of the site-directedmutagenized HC-mAβScFv fusion protein. A predicted N-linkedglycosylation site was found in CDR2 of the VH of the anti-Aβ MAb; themutagenized site is underlined in the figure. The asparagine (N) residueat position 497 was changed to an alanine (A) residue, and this changeis called N497A. The amino acid sequence is deduced from the nucleotidesequence (SEQ ID NO: 23).

FIG. 18. Nucleotide sequence (SEQ ID NO: 25) of the site-directedmutagenized fusion gene comprised of the anti-Aβ ScFv cDNA fused to the3′-end of the cDNA encoding the chimeric HIRMAb heavy chain encoded bythe eukaryotic expression plasmid, pCD-HC-mAβScFv plasmid (FIG. 2). The‘AG’ nucleotides have been mutagenized to ‘GC’ nucleotides at positions1552-1553; the site is underlined in the figure. This change innucleotide sequence results in the S499A amino acid change in the secondCDR of the VH of the mAbScFv, as shown in FIG. 19.

FIG. 19. Amino acid sequence (SEQ ID NO: 26) of the site-directedmutagenized HC-mAβScFv fusion protein. A predicted N-linkedglycosylation site was found in CDR2 of the VH of the anti-Aβ MAb; themutagenized site is underlined in the figure. The serine (S) residue atposition 499 was changed to an alanine (A) residue, and this change iscalled S499A. The amino acid sequence is deduced from the nucleotidesequence (SEQ ID NO: 25).

FIG. 20. Nucleotide sequence (SEQ ID NO: 27) of the tandem vectorencoding the intact antibody fusion protein comprised of the chimericHIRMAb light chain (LC) and a fusion protein heavy chain (HC), where themAβScFv was fused to the chimeric HIRMAb heavy chain. The tandem vectorencodes for murine dihydrofolate reductase (DHFR). The individualexpression cassettes of the tandem vector are shown in FIG. 24. From the5′-end to the 3′-end, the LC gene, the HC fusion gene, and the DHFR geneare contained in 3 separate expression cassettes on the tandem vector;and the open reading frames of each of these 3 expression cassettes areunderlined in the figure.

FIG. 21. Amino acid sequence (SEQ ID NO: 28) of the IgG signal peptidefollowed by the chimeric HIRMAb heavy chain followed by the Ab ScFv. Theamino acid sequence is deduced from the nucleotide sequence in FIG. 20.The amino acid sequences of the CDR1, CDR2, and CDR3 of the anti-HIRMAbheavy chain VH are in bold font. The constant region glycosylation site,NST, is underlined.

FIG. 22. Amino acid sequence (SEQ ID NO: 29) of the light chain encodedby the tandem vector. The amino acid sequence is deduced from thenucleotide sequence in FIG. 20. The amino acid sequences of the CDR1,CDR2, and CDR3 of the anti-HIRMAb light chain VL are in bold font.

FIG. 23. Amino acid sequence (SEQ ID NO: 30) of the DHFR encoded by thetandem vector. The amino acid sequence is deduced from the nucleotidesequence in FIG. 20.

FIG. 24. Genetic engineering of the antibody fusion protein tandemvector. The cDNA encoding the mAβScFv is produced by PCR from thepCD-HC-mAβScFv-I2V plasmid, and ligated into the HpaI site of the tandemvector precursor. The tandem vector encodes the antibody fusion proteinshown in FIG. 26. CMV=cytomegalovirus; HIR-LC=light chain (LC) of theanti-human insulin receptor (HIR) MAb; HIR-HC=heavy chain (HC) of theanti-HIRMAb; pA=poly A transcription termination sequence SV40=SV40promoter; DHFR=dihydrofolate reductase.

FIG. 25. Amino acid sequence of the 28 domains of the fusion antibodyheavy chain (SEQ ID NO:22): (1) signal peptide, (2) framework region(FR) 1 of the heavy chain (HC) of the chimeric HIRMAb, (3)complementarity determining region (CDR) 1 of the HC of the chimericHIRMAb, (4) FR2 of the HC of the chimeric HIRMAb, (5) CDR2 of the HC ofthe chimeric HIRMAb, (6) FR3 of the HC of the chimeric HIRMAb, (7) CDR3of the HC of the chimeric HIRMAb, (8) FR4 of the HC of the chimericHIRMAb, (9) CH1 region of the HC of the chimeric HIRMAb, (10) hingeregion of the HC of the chimeric HIRMAb, (11) CH2 region of the HC ofthe chimeric HIRMAb; the constant region glycosylation site, NST(Asn-Ser-Thr) is underlined, (12) CH3 region of the HC of the chimericHIRMAb, (13) Ser-Ser linker between the CH3 region of the HC of thechimeric HIRMAb and the beginning of the mAβScFv, (14) FR1 of thevariable region of the HC (VH) of the mAβScFv, (15) CDR1 of the VH ofthe mAβScFv, (16) FR2 of the VH of the mAβScFv, (17) CDR2 of the VH ofthe mAβScFv, (18) FR3 of the VH of the mAβScFv, (19) CDR3 of the VH ofthe mAβScFv, (20) FR4 of the VH of the mAβScFv, (21) 17 amino acidlinker between the VH and the variable region of the light chain (VL) ofthe mAβScFv, (22) FR1 of the VL of the mAβScFv b, (23) CDR1 of the VL ofthe mAβScFv, (24) FR2 of the VL of the mAβScFv, (25) CDR2 of the VL ofthe mAβScFv, (26) FR3 of the VL of the mAβScFv, (27) CDR3 of the VL ofthe mAβScFv, (28) FR4 of the VLH of the mAβScFv.

FIG. 26. Antibody fusion protein with 3 functionalities: (1) The CDRs ofthe chimeric HIRMAb bind to the BBB HIR to enable influx of the moleculefrom blood into brain across the BBB; (2) the CH2/CH3 interface of theFc region binds to the FcR receptor to enable efflux of the moleculefrom brain back to blood across the BBB; (3) the mAβScFv fused to theCH3 region binds to the Aβ amyloid peptide of AD to cause clearance ofamyloid from brain in AD. The role that each of these 3 functionalitiesplays in the clearance of brain amyloid in AD is shown in FIG. 27.

FIG. 27. The fusion protein clears amyloid from brain in AD via 3sequential steps, and each of these 3 sequential steps uses 3 separateparts of the antibody fusion protein molecule as shown in FIG. 26. Step1 is the influx of the fusion antibody from blood to brain across theBBB, which is mediated by binding of the fusion antibody to the BBBhuman insulin receptor (HIR). Step 2 is binding of the fusion antibodyto the amyloid plaque in AD, which promotes disaggregation of theamyloid plaque, and this binding to the plaque is mediated by themAβScFv part of the fusion antibody. Step 3 is the efflux of the fusionantibody from brain to blood across the BBB, which is mediated bybinding of the fusion antibody to the BBB FcR receptor at the CH2-CH3interface of the Fc region of the fusion antibody.

FIG. 28. Western blot with 9E10 MAb to C-terminal c-myc epitope of thesingle chain Fv (ScFv) antibody. (Lane 1): Fusion protein (45 kDa) ofstreptavidin (SA) and OX26 ScFv, used as a positive control. This OX26ScFv-SA fusion protein is comprised of 3 domains: OX26 ScFv, SA, andc-myc C-terminal epitope, and was affinity purified from bacterialpellets. (Lane 2): Negative control: media conditioned by COS cellstransfected with Lipofectamine and no plasmid DNA. The 9E10 anti-c-mycMAb cross-reacts with 2 proteins of 35-37 kDa that are secreted by COScells. Note the absence of the 29 kDa anti-Aβ ScFv in the media of COScells not transfected with pCD-mAβScFv. (Lane 3): Anti-Aβ ScFv obtainedfrom media conditioned by COS cells transfected with Lipofectamine 2000and pCD mAβScFv (FIG. 1B); the anti-Aβ ScFv has a molecular weight of 29kDa, and is 16 kDa smaller in size than the OX26 ScFv-SA fusion proteinin lane 1, owing to the presence of the 16 kDa SA moiety in the OX26ScFv/SA fusion protein.

FIG. 29. (A) Structure of ELISA used to demonstrate binding of mAβScFvto Aβ¹⁻⁴⁰ peptide, which is plated on a solid support. The mAβScFv bindsto the amyloid peptide, and the 9E10 anti-c-myc MAb binds to theEQKLISEEDL (SEQ ID NO:66) epitope fused to the carboxyl terminus of theprotein (FIG. 9). The 9E10 MAb is biotinylated, which enables detectionwith a conjugate of streptavidin and peroxidase. Medium conditioned byCOS cells that were transfected with pCD-mAβScFv produced a high signalin the assay, whereas medium from COS cells exposed only toLipofectamine (LIPO) produced no signal. The assay shows that themAβScFv binds to the Aβ¹⁻⁴⁰ peptide.

FIG. 30. Immunocytochemistry of frozen sections of AD autopsy brain,which were immune stained with medium conditioned by COS cells whichwere transfected with pCD-mAβScFv and expressing the mAβScFv (panels Aand C), with medium conditioned by COS cells which were exposed only toLipofectamine 2000 (panel B), or with the mouse IgG1 isotype controlantibody (panel D). Magnification bar in panels A and B is 88 um;magnification bar in panels C and D is 35 um. The assay shows that themAβScFv binds to the Aβ amyloid plaque of AD.

FIG. 31. Sodium dodecylsulfate polyacrylamide gel electrophoresis(SDS-PAGE) of protein A purified fusion antibody or chimeric HIRMAbunder reducing conditions, and stained with Coomasie blue. The gel showsa side by side comparison of the sizes of the heavy and light chains ofthe chimeric HIRMAb, and the fusion antibody. Both are comprised of thesame light chain, which is 28 kDa. The size of the heavy chain ofchimeric HIRMAb is 55 kDa, whereas the size of the heavy chain of fusionantibody is 82 kDa. The heavy chain of the fusion antibody includes the55 kDa heavy chain of chimeric HIRMAb fused to the 27 kDa anti-Aβ ScFv.

FIG. 32. Western blotting of protein A purified fusion antibody with ananti-human IgG primary antibody shows that the fusion antibody expressedin COS cells is processed and secreted intact with the expectedmolecular size. The blot shows a side by side comparison of theimmunoreactivity of the chimeric HIRMAb, and the fusion antibody. Bothare comprised of the same light chain, which is 28 kDa, as shown in theWestern blot. The size of the heavy chain of chimeric HIRMAb is 55 kDa,whereas the size of the heavy chain of fusion antibody is 82 kDa. Theheavy chain of the fusion antibody includes the 55 kDa heavy chain ofchimeric HIRMAb fused to the 27 kDa anti-Aβ ScFv.

FIG. 33. Isoelectric focusing of isoelectric standards, the chimericHIRMAb, the hybridoma generated murine anti-Aβ antibody, and the fusionantibody.

FIG. 34. Aβ¹⁻⁴⁰ immunoradiometric assay (IRMA) measures the binding ofthe [¹²⁵I]-murine anti-Aβ MAb to Aβ¹⁻⁴⁰ that is plated in wells of a96-well plate. The murine anti-Aβ MAb is the original murine MAb againstAβ purified from hybridoma conditioned medium. In the absence ofcompetitors of binding, approximately 20% of the total [¹²⁵I]-anti-AβMAb is bound to the Aβ¹⁻⁴⁰. Both the murine anti-Aβ MAb and the fusionantibody bind to the Aβ¹⁻⁴⁰, and the half saturation constant, K_(D), isnot significantly different. The affinity of the fusion antibody forAβ¹⁻⁴⁰ is equal to the affinity of the original murine anti-Aβ¹⁻⁴⁰ MAb

FIG. 35. The affinity of the chimeric HIRMAb, and the fusion antibodyfor the human insulin receptor (HIR) as determined by competitive ELISA,where the solid phase antigen is the affinity purified HIR extracellulardomain (ECD) produced from CHO cells. The avidity for the HIR ECD of thefusion antibody, ED₅₀=1.0±0.1 nM, is comparable to that of the chimericHIRMAb, ED₅₀=0.53±0.02 nM. There is no binding of human IgG1, theisotype control, to the HIR ECD.

FIG. 36. Binding of fusion protein to the HIR on the human BBB. (A)Isolated capillaries are purified from human autopsy brain, and used asan in vitro model of binding to the human BBB HIR. (B) Specific bindingof [¹²⁵I]-fusion antibody to human brain capillaries is time-dependent,whereas binding of the [¹²⁵I]-mouse anti-Aβ antibody is constant withtime, and is non-specific.

FIG. 37. In vivo pharmacokinetics in adult Rhesus monkey. The[¹²⁵I]-fusion antibody, and the [³H]-mouse anti-Aβ antibody, wereinjected intravenously into the adult Rhesus monkey and serumconcentrations [% of injected dose (I.D.)/mL] determined over a 3 hourperiod. There is no measurable clearance from blood of the [¹²⁵I]-mouseanti-Aβ antibody during this time period, whereas the fusion antibody iscleared from serum.

FIG. 38. Brain volume of distribution (VD) of the [³H]-mouse anti-Aβantibody and the [¹²⁵I]-fusion antibody in Rhesus monkey brain at 3hours after a single intravenous injection of both labeled antibodies.The VD for both the homogenate and the post-vascular supernatant isshown. The VD for the [³H]-mouse anti-Aβ antibody, 10 uL/g, is equal tothe brain blood volume, and indicates this antibody is not transportedacross the primate BBB in vivo. The VD for the [¹²⁵I]-fusion antibodyis >10-fold higher than for the [³H]-mouse anti-Aβ antibody, in both thebrain homogenate and the post-vascular supernatant, which indicates the[¹²⁵I]-fusion antibody is transported across the BBB from blood tobrain.

FIG. 39. Brain scans of adult Rhesus monkey at 3 hours after theintravenous administration of the [¹²⁵I]-fusion antibody demonstrateswidespread distribution of the fusion antibody into the primate brain invivo from blood. The top scan is the most frontal part of brain, and thebottom scan is the most caudal part of brain, and includes thecerebellum.

FIG. 40. Efflux from brain to blood of the [¹²⁵I]-fusion antibody in theadult rat. The [¹²⁵I]-fusion antibody was injected into the cortex understereotaxic guidance, and the efflux of the fusion antibody from brainacross the BBB was measured at 90 minutes after the injection. At thistime nearly 60% of the injected fusion antibody had effluxed from brain.This efflux was completely blocked by the co-injection of human Fcfragments, which indicates the efflux is mediated by a Fc receptor atthe BBB.

FIG. 41. (A) Outline of Aβ plaque disaggregation assay. The secondaryantibody is an anti-human IgG for study of the fusion antibody, and isan anti-mouse IgG for study of the mouse anti-Aβ MAb. (B, C)Disaggreagation of Aβ amyloid in vitro by the fusion antibody (B) or bythe mouse anti-Aβ MAb (C). Aβ¹⁻⁴⁰ aggregates were formed over 6 days,followed by incubation with the fusion antibody, with human IgG1(hIgG1), or with phosphate buffered saline (PBS) for either 1 or 4 hoursat 37 C (B), or with the mouse anti-Aβ antibody, with non-immune mouseIgG, or PBS for either 1 or 4 hours at 37 C (C). In parallel, anantibody that binds to the carboxyl terminal region of the Aβ¹⁻⁴⁰peptide was plated in 96 well plates, as depicted in panel A. Theanti-Aβ ScFv portion of the fusion antibody, or the mouse anti-Aβ MAb,binds to the amino terminal part of Aβ¹⁻⁴⁰. Therefore, a positive ELISAsignal is generated only if plaque is present. The data show that thefusion antibody, and the mouse anti-Aβ MAb, selectively bind to Aβ¹⁻⁴⁰plaque, and that this binding causes disaggregation over a 4 hourperiod.

FIG. 42. (A) Film autoradiography of AD autopsy brain sections labeledwith [¹²⁵I]-fusion antibody, showing binding of antibody to vascularamyloid plaque. (B) Immunocytochemistry of AD autopsy brain sectionslabeled with murine anti-Aβ MAb showing antibody binding to vascularamyloid of AD.

FIG. 43. Conjugation of1,4,7,10-tetraazacyclododecane-N,—N′,N″,N′″-tetraacetic acid (DOTA) tothe HIRMAb does not inhibit binding of the antibody to the HIRextracellular domain, as shown by the HIR ELISA. The DOTA is a highaffinity chelator of radionuclide metals, such as 111-indium, andenables production of a radiopharmaceutical for neurodiagnosis and brainscanning with the DOTA conjugated fusion antibody.

FIG. 44. The anti-WNV antibody (Ab) does not cross the blood-brainbarrier (BBB). However, the anti-WNV Ab can cross the BBB followingfusion to a molecular Trojan horse (TH), which is itself anotherantibody to the insulin receptor (IR). The TH undergoesreceptor-mediated transport across the BBB via transport on theendogenous IR, and carries the anti-WNV Ab into the brain, where the Abcan neutralize the virus. The TH is also a ligand for the BBB Fcreceptor (FcR), which allows for net export of the WNV from brain.

FIG. 45. Ethidium bromide stain of agarose gel electrophoresis of thePCR reaction following amplification of the anti-WNV VH (A), theanti-WNV VL (B), and the DIII (C). MW size standards on shown on theright. The VH, VL, and DIII cDNAs were produced by PCR with the ODNsshown in Tables 4, 5, and 6, respectively.

FIG. 46. Genetic engineering of COS cell expression plasmids fortransient expression of anti-WNV ScFv, from pCD-WNV-ScFv, and anti-BSAheavy chain, from pCD-HC-BSA, respectively. The pCD-ScFv vector is anScFv expression vector that enables fusion of a VH and VL via anintermediate 17-amino acid linker. 6×His tag disclosed as SEQ ID NO: 68.

FIG. 47. Amino acid sequence of the heavy chain of the anti-WNV-fusionantibody (SEQ ID NO: 65), which is comprised of the following domains:(i) IgG signal peptide, (ii) HIRMAb heavy chain variable region (VH),(iii) human IgG1 constant region, which is comprised of 4 sub-domains:CH1, hinge, CH2, and CH3, (iv) linker separating the HIRMAb heavy chainand the anti-WNV ScFv, (v) the anti-WNV ScFv heavy chain variable region(VH), (vi) a 17 amino acid linker separating the ScFv VH and VL, and(vii) the anti-WNV ScFv light chain variable region (VL). Thecomplementarity determining regions (CDRs) of the HIRMAb and theanti-WNV antibody are indicated in the figure.

FIG. 48. Genetic engineering of CHO cell tandem vector for expression ofthe anti-WNV fusion antibody (BSA) from 3 precursor plasmids: the fusionheavy chain plasmid, pCD-HC-BSA, the light chain plasmid, pCD-LC, andthe wild type (wt) dihydrofolate reductase (DHFR) plasmid, pwt DHFR.

DETAILED DESCRIPTION OF THE INVENTION

The blood-brain barrier is a limiting factor in the delivery of manyperipherally-administered agents to the central nervous system,including therapeutic or diagnostic monoclonal antibodies. The presentinvention addresses three factors that are important in delivering acomposition, e.g., an antibody composition such as a pharmaceutical ordiagnostic, across the BBB to the CNS: 1) Modification of thecomposition, e.g., antibody to allow it to influx across the BBB in theblood to brain direction; 2) Modification of the composition, e.g.,antibody to allow it to efflux across the BBB in the brain to blooddirection; and 3) Retention of activity of the composition, e.g.,antibody once across the BBB and in brain. Various aspects of theinvention address these factors, by providing fusion structures (e.g.,fusion proteins) of antibody pharmaceutical covalently linked to one ormore structures that causes the agent to be transported across the BBBin one or both directions, and/or to retain some or all of its activityin the brain while still attached to the structure.

Accordingly, in one aspect provided herein is a composition of molecularweight greater than about 1000 Daltons that is capable of crossing theBBB from the blood to the brain; and crossing the BBB from the brain tothe blood.

In another aspect provided herein is a method of diagnosing a CNSdisorder that includes measuring the level of a composition in a bodyfluid of an individual, where the composition is capable of crossing theBBB from the blood to the brain, interacting with a pathologicalsubstance associated with a brain disorder, and crossing the BBB fromthe brain to the blood, and where the composition has been administeredto the individual and has interacted with the pathological substance.

In a further aspect provided herein is a method of treating Alzheimer'sdisease comprising systemically administering a composition comprising afirst portion that crosses the BBB from the blood to the brain via afirst receptor-mediated BBB; a second portion that crosses the BBB fromthe brain to the blood via a second receptor-mediated BBB transportsystem; and a third portion that interacts with Aβ peptide.

A listing of the abbreviations of terms used herein (e.g., orf, PrP, andScFv) is provided at the end of the Detailed Description section.

As used herein, an “agent” includes any substance that is useful inproducing an effect, including a physiological or biochemical effect inan organism. A “therapeutic agent” is a substance that produces or isintended to produce a therapeutic effect, i.e., an effect that leads toamelioration, prevention, and/or complete or partial cure of a disorder.A “therapeutic effect,” as that term is used herein, also includes theproduction of a condition that is better than the average or normalcondition in an individual that is not suffering from a disorder, i.e.,a supranormal effect such as improved cognition, memory, mood, or othercharacteristic attributable at least in part to the functioning of theCNS, compared to the normal or average state. An “antibody therapeuticagent” is an antibody that produces a therapeutic effect in the CNS.

As used herein, an “antibody that is active in the central nervoussystem (CNS)” includes antibodies that have an effect when administeredto the CNS. The effect may be a therapeutic effect or a non-therapeuticeffect, e.g., a diagnostic effect or an effect useful in research. Ifthe effect is a therapeutic effect, then the antibody is also atherapeutic antibody. If the effect is a diagnostic effect, then theantibody is also a diagnostic antibody. An antibody may besimultaneously a diagnostic and a therapeutic antibody.

As used herein, “capable of” (e.g., “capable of crossing the BBB”)refers to an operational functional property of a structure (e.g.,protein, antibody, compound, or composition) conferred by one or morestructural features (i.e., intrinsic features) of the structure.

“Treatment” or “treating” as used herein includes achieving atherapeutic benefit and/or a prophylactic benefit. By therapeuticbenefit is meant eradication or amelioration of the underlying disorderor condition being treated. For example, in an individual with aneurological disorder, therapeutic benefit includes partial or completehalting of the progression of the disorder, or partial or completereversal of the disorder. Also, a therapeutic benefit is achieved withthe eradication or amelioration of one or more of the physiological orpsychological symptoms associated with the underlying condition suchthat an improvement is observed in the patient, notwithstanding the factthat the patient may still be affected by the condition. A prophylacticbenefit of treatment includes prevention of a condition, retarding theprogress of a condition (e.g., slowing the progression of a neurologicaldisorder), or decreasing the likelihood of occurrence of a condition. Asused herein, “treating” or “treatment” includes prophylaxis.

As used herein, the term “effective amount” can be an amount sufficientto effect beneficial or desired results, such as beneficial or desiredclinical results, or enhanced cognition, memory, mood, or other desiredCNS results. An effective amount is also an amount that produces aprophylactic effect, e.g., an amount that delays, reduces, or eliminatesthe appearance of a pathological or undesired condition. Such conditionsof the CNS include dementia, neurodegenerative diseases as describedherein, suboptimal memory or cognition, mood disorders, general CNSaging, or other undesirable conditions. An effective amount can beadministered in one or more administrations. In terms of treatment, an“effective amount” of a composition of the invention is an amount thatis sufficient to palliate, ameliorate, stabilize, reverse or slow theprogression of a disorder, e.g., a neurological disorder. An “effectiveamount” may be of any of the compositions of the invention used alone orin conjunction with one or more agents used to treat a disease ordisorder. An “effective amount” of a therapeutic agent within themeaning of the present invention will be determined by a patient'sattending physician or veterinarian. Such amounts are readilyascertained by one of ordinary skill in the art and will a therapeuticeffect when administered in accordance with the present invention.Factors which influence what a therapeutically effective amount will beinclude, the specific activity of the therapeutic agent being used, thetype of disorder (e.g., acute vs. chronic neurological disorder), timeelapsed since the initiation of the disorder, and the age, physicalcondition, existence of other disease states, and nutritional status ofthe individual being treated. Additionally, other medication the patientmay be receiving will affect the determination of the therapeuticallyeffective amount of the therapeutic agent to administer.

A “subject” or an “individual,” as used herein, is an animal, forexample, a mammal. In some embodiments a “subject” or an “individual” isa human. In some embodiments, the subject suffers from a neurologicaldisorder.

In some embodiments, an agent is “administered peripherally” or“peripherally administered.” As used herein, these terms refer to anyform of administration of an agent, e.g., a therapeutic antibody, to anindividual that is not direct administration to the CNS, i.e., thatbrings the agent in contact with the non-brain side of the blood-brainbarrier. “Peripheral administration,” as used herein, includesintravenous, subcutaneous, intramuscular, intraperitoneal, transdermal,inhalation, transbuccal, intranasal, rectal, and oral administration.

A “pharmaceutically acceptable carrier” or “pharmaceutically acceptableexcipient” herein refers to any carrier that does not itself induce theproduction of antibodies harmful to the individual receiving thecomposition. Such carriers are well known to those of ordinary skill inthe art. A thorough discussion of pharmaceutically acceptablecarriers/excipients can be found in Remington's Pharmaceutical Sciences,Gennaro, A R, ed., 20th edition, 2000: Williams and Wilkins PA, USA.Exemplary pharmaceutically acceptable carriers can include salts, forexample, mineral acid salts such as hydrochlorides, hydrobromides,phosphates, sulfates, and the like; and the salts of organic acids suchas acetates, propionates, malonates, benzoates, and the like. Forexample, compositions of the invention may be provided in liquid form,and formulated in saline based aqueous solution of varying pH (5-8),with or without detergents such polysorbate-80 at 0.01-1%, orcarbohydrate additives, such mannitol, sorbitol, or trehalose. Commonlyused buffers include histidine, acetate, phosphate, or citrate.

A “recombinant host cell” or “host cell” refers to a cell that includesan exogenous polynucleotide, regardless of the method used forinsertion, for example, direct uptake, transduction, f-mating, or othermethods known in the art to create recombinant host cells. The exogenouspolynucleotide may be maintained as a nonintegrated vector, for example,a plasmid, or alternatively, may be integrated into the host genome.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues.That is, a description directed to a polypeptide applies equally to adescription of a peptide and a description of a protein, and vice versa.The terms apply to naturally occurring amino acid polymers as well asamino acid polymers in which one or more amino acid residues is anon-naturally occurring amino acid, e.g., an amino acid analog. As usedherein, the terms encompass amino acid chains of any length, includingfull length proteins (i.e., antigens), wherein the amino acid residuesare linked by covalent peptide bonds.

The term “amino acid” refers to naturally occurring and non-naturallyoccurring amino acids, as well as amino acid analogs and amino acidmimetics that function in a manner similar to the naturally occurringamino acids. Naturally encoded amino acids are the 20 common amino acids(alanine, arginine, asparagine, aspartic acid, cysteine, glutamine,glutamic acid, glycine, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, proline, serine, threonine, tryptophan,tyrosine, and valine) and pyrolysine and selenocysteine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an α carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, such as,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (such as, norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

The term “nucleic acid” refers to deoxyribonucleotides,deoxyribonucleosides, ribonucleosides, or ribonucleotides and polymersthereof in either single- or double-stranded form. Unless specificallylimited, the term encompasses nucleic acids containing known analoguesof natural nucleotides which have similar binding properties as thereference nucleic acid and are metabolized in a manner similar tonaturally occurring nucleotides. Unless specifically limited otherwise,the term also refers to oligonucleotide analogs including PNA (peptidenucleic acid), analogs of DNA used in antisense technology(phosphorothioates, phosphoroamidates, and the like). Unless otherwiseindicated, a particular nucleic acid sequence also implicitlyencompasses conservatively modified variants thereof (including but notlimited to, degenerate codon substitutions) and complementary sequencesas well as the sequence explicitly indicated. Specifically, degeneratecodon substitutions may be achieved by generating sequences in which thethird position of one or more selected (or all) codons is substitutedwith mixed-base and/or deoxyinosine residues (Batzer et al., NucleicAcid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608(1985); and Cassol et al. (1992); Rossolini et al., Mol. Cell. Probes8:91-98 (1994)).

The terms “isolated” and “purified” refer to a material that issubstantially or essentially removed from or concentrated in its naturalenvironment. For example, an isolated nucleic acid may be one that isseparated from the nucleic acids that normally flank it or other nucleicacids or components (proteins, lipids, etc. . . . ) in a sample. Inanother example, a polypeptide is purified if it is substantiallyremoved from or concentrated in its natural environment. Methods forpurification and isolation of nucleic acids and peptides are well knownin the art.

In one aspect, the invention provides compositions and methods thatutilize an agent, e.g., an antibody, covalently linked to a structurecapable of crossing the blood-brain barrier (BBB). The compositions andmethods are useful in transporting agents, e.g. antibodies, from theperipheral blood and across the blood-brain barrier into the CNS. Inaddition, in some aspects, compositions and methods of the inventionutilize structures that are further capable of crossing the BBB from theCNS to the blood. As used herein, the “blood-brain barrier” refers tothe barrier between the peripheral circulation and the brain and spinalcord which is formed by tight junctions within the brain capillaryendothelial plasma membranes, creates an extremely tight barrier thatrestricts the transport of molecules into the brain, even molecules assmall as urea, molecular weight of 60 Da. The blood-brain barrier withinthe brain, the blood-spinal cord barrier within the spinal cord, and theblood-retinal barrier within the retina, are contiguous capillarybarriers within the central nervous system (CNS), and are collectivelyreferred to herein as the blood-brain barrier or BBB.

The BBB is a limiting step in the development of new neurotherapeutics,diagnostics, and research tools for the brain and CNS. Essentially 100%of large molecule therapeutics such as recombinant proteins, antisensedrugs, gene medicines, monoclonal antibodies, or RNA interference(RNAi)-based drugs, do not cross the BBB in pharmacologicallysignificant amounts. While it is generally assumed that small moleculedrugs can cross the BBB, in fact, <2% of all small molecule drugs areactive in the brain owing to the lack transport across the BBB. Amolecule must be lipid soluble and have a molecular weight less than 400Daltons (Da) in order to cross the BBB in pharmacologically significantamounts, and the vast majority of small molecules do not have these dualmolecular characteristics. Therefore, most potentially therapeutic,diagnostic, or research molecules do not cross the BBB inpharmacologically active amounts. So as to bypass the BBB, invasivetranscranial drug delivery strategies are used, such asintracerebro-ventricular (ICV) infusion, intracerebral (IC)administration, and convection enhanced diffusion (CED). Transcranialdrug delivery to the brain is expensive, invasive, and largelyineffective. The ICV route delivers therapeutic proteins such asantibody pharmaceuticals only to the ependymal surface of the brain, notinto brain parenchyma, which is typical for drugs given by the ICVroute. The IC administration of a pharmaceutical only delivers drug tothe local injection site, owing to the low efficiency of drug diffusionwithin the brain. The CED of pharmaceuticals results in preferentialfluid flow through the white matter tracts of brain, which can lead todemyelination, and astrogliosis.

The present invention offers an alternative to these highly invasive andgenerally unsatisfactory methods for bypassing the BBB, allowingmacromolecular agents, e.g., antibody pharmaceuticals to cross the BBBfrom the peripheral blood and, in some embodiments, allowing the agentor the agent in modified form (e.g., antibody bound to antigen) to crossthe BBB from the brain to the blood. Without wishing to be bound bytheory, it is thought that it is based on the use of endogenoustransport systems present in the BBB to provide a mechanism to transporta desired substance from the peripheral blood to the CNS.

For example, in one aspect, the invention provides a composition ofmolecular weight greater than about 1000 Daltons that is capable ofcrossing the BBB from the blood to the brain and crossing the BBB fromthe brain to the blood.

Compositions of MW Greater than about 1000 Daltons Capable of Crossingthe BBB in Both Directions

In one aspect, the invention provides composition of molecular weightgreater than about 1000 Daltons that is capable of (i) crossing the BBBfrom the blood to the brain; and (ii) crossing the BBB from the brain tothe blood. In some embodiments, the composition is further capable ofinteracting with a substance in the brain, e.g., a pathologicalsubstance associated with a brain disorder such as Alzheimer's disease,Parkinson's disease, Huntington's disease, bovine spongiformencephalopathy, West Nile virus encephalitis, Neuro-AIDS, brain injury,spinal cord injury, metastatic cancer of the brain, metastatic breastcancer of the brain, primary cancer of the brain, or multiple sclerosis.The pathological substance can be of a type selected from the groupconsisting of proteins, nucleic acids, carbohydrates, carbohydratepolymers, lipids, glycolipids, small molecules, and combinationsthereof. In some embodiments, the pathological substance is a protein,such as Aβ amyloid, α-synuclein, huntingtin Protein, PrP prion protein,West Nile envelope protein, tumor necrosis factor (TNF) relatedapoptosis inducing ligand (TRAIL), Nogo A, HER2, epidermal growth factorreceptor (EGFR), hepatocyte growth factor (HGF), or oligodendrocytesurface antigen. In some embodiments, the protein is Aβ amyloid.

In some embodiments, the invention provides compositions that include astructure that binds to a BBB receptor mediated transport system. Thestructure may be coupled to an active agent, e.g., an antibodypharmaceutical, diagnostic, or research moiety, for which transportacross the BBB is desired, e.g., a neurotherapeutic agent. Thecompositions and methods of the invention may utilize any suitablestructure that is capable of transport by the selected endogenous BBBreceptor-mediated transport system, and that is also capable ofattachment to the desired agent, e.g., antibody. In some embodiments,the targeting structure is itself an antibody. In some embodiment thetargeting antibody is a monoclonal antibody (MAb), e.g., a chimeric MAb.

Endogenous BBB Receptor-Mediated Transport Systems

The BBB has been shown to have specific receptors that allow thetransport from the blood to the brain of several macromolecules; thesetransporters are suitable as transporters for compositions of theinvention. Endogenous BBB receptor-mediated transport systems useful inthe invention include, but are not limited to, those that transportinsulin, transferrin, insulin-like growth factors 1 and 2 (IGF1 andIGF2), leptin, and lipoproteins. In some embodiments, the inventionutilizes a structure that is capable of crossing the BBB via theendogenous insulin BBB receptor-mediated transport system, e.g., thehuman endogenous insulin BBB receptor-mediated transport system.

One noninvasive approach for the delivery of agents to the CNS, and, insome embodiments, transport out of the CNS, is to attach the agent ofinterest to a structure, e.g., molecule that binds with receptors on theBBB. The structure then serves as a vector for transport of the agentacross the BBB. Such structures are referred to herein as “molecularTrojan horses (MTH).” Typically, though not necessarily, a MTH is anexogenous peptide or peptidomimetic moiety (e.g., a MAb) capable ofbinding to an endogenous BBB receptor mediated transport system thattraverses the BBB on the endogenous BBB receptor-mediated transportsystem. In certain embodiments, the MTH can be an antibody to a receptorof the transport system, e.g., the insulin receptor. In someembodiments, the antibody is a monoclonal antibody (MAb). In someembodiments, the MAb is a chimeric MAb. Thus, despite the fact thatantibodies in blood are normally are excluded from the brain, they canbe an effective vehicle for the delivery of molecules into the brainparenchyma if they have specificity for receptors on the BBB.

Accordingly, antibodies are particularly useful in embodiments of theinvention, especially MAbs. Certain receptor-specific MAbs may mimic theendogenous ligand and function as a MTH and traverse a plasma membranebarrier via transport on the specific receptor system. In certainembodiments, the MTH is a MAb to the human insulin receptor (HIR) on thehuman BBB. The HIR MAb binds an exofacial epitope on the human BBB HIRand this binding enables the MAb to traverse the BBB via a transportreaction that is mediated by the human BBB insulin receptor.

For use in humans, a chimeric HIR Ab is preferred that contains enoughhuman sequence that it is not significantly immunogenic whenadministered to humans, e.g., about 80% human and about 20% mouse, orabout 85% human and about 15% mouse, or about 90% human and about 10%mouse, or about 95% human and 5% mouse, or greater than about 95% humanand less than about 5% mouse. Chimeric antibodies to the human BBBinsulin receptor with sufficient human sequences for use in theinvention are described in, e.g., Coloma et al. (2000) Pharm. Res. 17:266-274, which is incorporated by reference herein in its entirety. Amore highly humanized form of the HIR MAb can also be engineered, andthe humanized HIRMAb has activity comparable to the murine HIRMAb andcan be used in embodiments of the invention. See, e.g., U.S. PatentApplication Publication No. 20040101904, filed Nov. 27, 2002,incorporated by reference herein in its entirety.

Current technologies permit a vast number of sequence variants ofcandidate HIR Abs or known HIR Abs to be readily generated be (e.g., invitro) and screened for binding to a target antigen such as the ECD ofthe human insulin receptor or an isolated epitope thereof. See, e.g.,Fukuda et al. (2006) “In vitro evolution of single-chain antibodiesusing mRNA display,” Nuc. Acid Res., 34(19) (published online) for anexample of ultra high throughput screening of antibody sequencevariants. See also, Chen et al. (1999), “In vitro scanning saturationmutagenesis of all the specificity determining residues in an antibodybinding site,” Prot Eng, 12(4): 349-356. An insulin receptor ECD can bepurified as described in, e.g., Coloma et al. (2000) Pharm Res,17:266-274, and used to screen for HIR Abs and HIR Ab sequence variantsof known HIR Abs.

An “antibody” or “antibody construct,” as those term are used herein,includes reference to any molecule, whether naturally-occurring,artificially induced, or recombinant, which has specific immunoreactiveactivity. Generally, though not necessarily, an antibody is a proteinthat includes two molecules, each molecule having two differentpolypeptides, the shorter of which functions as the light chains of theantibody and the longer of which polypeptides function as the heavychains of the antibody. Normally, as used herein, an antibody willinclude at least one variable region from a heavy or light chain.Additionally, the antibody may comprise combinations of variableregions. The combination may include more than one variable region of alight chain or of a heavy chain. The antibody may also include variableregions from one or more light chains in combination with variableregions of one or more heavy chains. An antibody can be animmunoglobulin molecule obtained by in vitro or in vivo generation ofthe humoral response, and includes both polyclonal and monoclonalantibodies. Furthermore, the present invention includes antigen bindingfragments of the antibodies described herein, such as Fab, Fab′, F(ab)₂,and Fv fragments, fragments comprised of one or more CDRs, single-chainantibodies (e.g., single chain Fv fragments (ScFv)), disulfidestabilized (dsFv) Fv fragments, heteroconjugate antibodies (e.g.,bispecific antibodies), pFv fragments, heavy chain monomers or dimers,light chain monomers or dimers, and dimers consisting of one heavy chainand one light chain, all of which are encompassed by the terms“antibody” or “antibody structure.” Such antibody fragments may beproduced by chemical methods, e.g., by cleaving an intact antibody witha protease, such as pepsin or papain, or via recombinant DNA techniques,e.g., by using host cells transformed with truncated heavy and/or lightchain genes. Synthetic methods of generating such fragments are alsocontemplated. Heavy and light chain monomers may similarly be producedby treating an intact antibody with a reducing agent, such asdithiothreitol or .beta.-mercaptoethanol, or by using host cellstransformed with DNA encoding either the desired heavy chain or lightchain or both. An antibody immunologically reactive with a particularantigen can be generated in vivo or by recombinant methods such asselection of libraries of recombinant antibodies in phage or similarvectors.

A “chimeric” antibody includes an antibody derived from a combination ofdifferent mammals. The mammal may be, for example, a rabbit, a mouse, arat, a goat, or a human. The combination of different mammals includescombinations of fragments from human and mouse sources.

In some embodiments, an antibody of the present invention is amonoclonal antibody (MAb), typically a human monoclonal antibody. Suchantibodies are obtained from transgenic mice that have been “engineered”to produce specific human antibodies in response to antigenic challenge.In this technique, elements of the human heavy and light chain locus areintroduced into strains of mice derived from embryonic stem cell linesthat contain targeted disruptions of the endogenous heavy chain andlight chain loci. The transgenic mice can synthesize human antibodiesspecific for human antigens, and the mice can be used to produce humanantibody-secreting hybridomas.

Antibodies used in the invention may be glycosylated ornon-glycosylated. If the antibody is glycosylated, any pattern ofglycosylation that does not significantly affect the function of theantibody may be used. Glycosylation can occur in the pattern typical ofthe cell in which the antibody is made, and may vary from cell type tocell type. For example, the glycosylation pattern of a monoclonalantibody produced by a mouse myeloma cell can be different than theglycosylation pattern of a monoclonal antibody produced by a transfectedChinese hamster ovary (CHO) cell. In some embodiments, the antibody isglycosylated in the pattern produced by a transfected Chinese hamsterovary (CHO) cell.

Accordingly, in some embodiments, a genetically engineered HIR MAb, withthe desired level of human sequences, is fused to an agent for whichtransport across the BBB is desired, e.g. an antibody pharmaceutical, toproduce a recombinant fusion protein that is a bi-functional molecule.The recombinant therapeutic antibody/HIRMAb is able to (i) cross thehuman BBB in the blood to brain direction, via transport on the BBB HIR,(ii) cross the human BBB in the brain to blood direction, via transporton the BBB FcR receptor, and (iii) bind the therapeutic antibody'starget, e.g., the Aβ amyloid of AD, to cause neurotherapeutic effectsonce inside the brain, following peripheral administration.

The invention provides compositions and methods for transport of agentsacross the BBB from blood to CNS and/or from CNS to blood. One usefulclass of agents is antibody pharmaceuticals, which include therapeuticand diagnostic antibody structures. The antibody pharmaceutical agentfor which transport across the BBB is desired may be any suitableantibody substance for introduction into the CNS. Generally, the agentis an antibody for which transport across the BBB is desired, which doesnot, in its native form, cross the BBB in significant amounts. Theantibody agent may be, e.g., a therapeutic agent, a diagnostic agent, ora research agent. Diagnostic agents include antibodyradiopharmaceuticals, such as an anti-Aβ antibody for imaging the Aβamyloid burden in the brain of patients with AD. In some embodiments,the antibody pharmaceutical is a therapeutic agent, such as antibodythat disaggregates the Aβ amyloid in the brain of patients with AD.Antibody agents useful in the invention are listed in Table 1.

TABLE 1 Treatment of Brain Disorders with Monoclonal AntibodyTherapeutics Target for Monoclonal Antibody Disease Aβ amyloidAlzheimer's disease α-synuclein Parkinson's disease Huntingtin proteinHuntington's disease PrP prion protein Mad cow disease West Nileenvelope protein West Nile virus encephalitis tumor necrosis factor(TNF) related Neuro-AIDS apoptosis inducing ligand (TRAIL) Nogo A Braininjury, spinal cord injury, stroke HER2 Metastatic breast cancer ofbrain epidermal growth factor receptor (EGFR); Primary and metastaticcancer hepatocyte growth factor (HGF) of brain Oligodendrocyte surfaceantigen Multiple sclerosis

One type of agent of use in the invention is antibody agents. Manyantibody agents, e.g., pharmaceuticals, are active (e.g.,pharmacologically active) in brain but do not cross the blood-brainbarrier. These factors are suitable for use in the compositions andmethods of the invention and include an antibody that is directedagainst the Aβ amyloid peptide of Alzheimer's disease (AD) for thediagnosis or treatment of AD. In some embodiments, the antibody isdirected against α-synuclein of Parkinson's disease (PD) for thediagnosis or treatment of PD. In some embodiments, the antibody isdirected against the huntingtin protein of Huntington's disease (HD) forthe diagnosis or treatment of HD. In some embodiments, the antibody isdirected against the Prp protein of scrapie or mad cow disease for thediagnosis or treatment of human equivalents of scrapie. In someembodiments, the antibody is directed against an envelope protein of theWest Nile virus for the diagnosis or treatment of West Nileencephalitis. In some embodiments, the antibody is directed against thetumor necrosis factor (TNF) related apoptosis inducing ligand (TRAIL)for the diagnosis or treatment of acquired immune deficiency syndrome(AIDS), which infects the brain. In some embodiments, the antibody isdirected against the nogo A protein for the diagnosis or treatment ofbrain injury, spinal cord injury, or stroke. In some embodiments, theantibody is directed against the HER2 protein for the diagnosis ortreatment of breast cancer metastatic to the brain. In some embodiments,the antibody is directed against oncogenic receptor proteins such as theepidermal growth factor receptor (EGFR) for the diagnosis or treatmentof either primary brain cancer or metastatic cancer of the brain. Insome embodiments, the antibody is directed against an oncogenic growthfactor such as the epidermal growth factor (EGF) or the hepatocytegrowth factor (HGF) for the diagnosis or treatment of either primarybrain cancer or metastatic cancer of the brain. In some embodiments, theantibody is directed against an oligodendrocyte surface antigen for thediagnosis or treatment of demyelinating disease such as multiplesclerosis. Particularly useful in some embodiments of the inventionutilizing ScFv forms of the antibody, e.g., therapeutic antibody, thatare used as precursors for fusion proteins that cross the BBB are thosethat naturally form dimeric structures, similar to original antibody.Some embodiments of the invention provides a fusion protein constructedof ScFv derived from the antibody fused to one chain (e.g., a light orheavy chain) of a targeting antibody, e.g., of the HIRMAb. Typically,the molecular weight range of antibodies that may be fused to themolecular Trojan horse ranges from 1000 Daltons to 500,000 Daltons.

One particularly useful antibody pharmaceutical in embodiments of theinvention is an antibody against the Aβ amyloid peptide of AD. Thedementia of AD is caused by the progressive accumulation over many yearsof amyloid plaque. This plaque is formed by the aggregation of the Aβamyloid peptide, which is a 40-43 amino acid peptide designatedAβ^(1-40/43), which is derived from the proteolytic processing withinthe brain of the amyloid peptide precursor protein called APP.

A potential therapy for AD is any drug that can enter the brain andcause disaggregation of the amyloid plaque. Transgenic mice have beenengineered which express mutant forms of the APP protein, and these micedevelop amyloid plaque similar to people with AD. The amyloid plaque canbe disaggregated with the application of anti-Aβ antibodies administereddirectly into the brain of the transgenic mice via either directcerebral injection or via a cranial window. Following anti-Aβantibody-mediated disaggregation of the amyloid plaque, the dystrophicnerve endings in the vicinity of the plaque begin to heal and formnormal structures. The anti-Aβ antibody must be injected directly intothe brain via needle because the antibody does not cross the BBB.Therefore, antibody administered in the blood cannot access the plaquein brain behind the BBB.

Antibody based therapies of AD include active or passive immunizationagainst the Aβ peptide. In active immunization, the subject is immunizedwith the Aβ peptide along with an adjuvant such as Freund's adjuvant.Active immunization of transgenic mice resulted in a decrease in theamyloid burden in brain, which is evidence that the anti-Aβ peptideantibodies in the blood formed in the active immunization treatment wereable to cross the BBB in the immunized mouse. It is well known that theadministration of adjuvants such as Freunds adjuvant causes disruptionof the BBB via an inflammatory response to the adjuvant administration.It is likely that active immunization in humans with AD will either notbe effective, because (a) the adjuvant used in humans is not toxic, andthe BBB is not disrupted, or (b) the adjuvant is toxic, and causesopening of the BBB via an inflammatory response to the adjuvant. Openingof the BBB allows the entry into brain of serum proteins such asalbumin, and these proteins are toxic to brain cells. In passiveimmunization, an anti-Aβ peptide antibody is administered directly tothe subject with brain amyloid, and this has been done in transgenicmice with brain amyloid similar to AD. However, the dose of anti-Aβpeptide antibody that must be administered to the mice is prohibitivelyhigh, owing to the lack of significant transport of antibody moleculesin the blood to brain direction. Therefore, the limiting factor ineither the active or passive immunization of either transgenic mice orof people with AD and brain amyloid is the BBB, and the lack oftransport of antibody molecules across the BBB in the blood to braindirection.

As used herein, the term “anti-Aβ peptide antibody” includes thepharmaceutically acceptable salts, polymorphs, hydrates, solvates,biologically-active fragments, biologically active variants andstereoisomers of the precursor anti-Aβ peptide antibody, as well asagonist, mimetic, and antagonist variants of antibodies directed atalternative targets, which cross-react with the anti-Aβ peptideantibody, and polypeptide fusion variants thereof. Variants include oneor more deletions, substitutions, or insertions in the sequence of theanti-Aβ peptide antibody precursor. When the targeting agent is also anantibody, e.g., a MAb such as HIRMAb is used, additional fusion proteinvariants can be produced with the substitution of amino acids withineither the framework region (FR) or the complementarity determiningregion (CDR) of either the light chain or the heavy chain of theantibody, e.g., HIRMAb, as long as the fusion protein binds with highaffinity to the endogenous receptor, e.g., HIR to promote transportacross the human BBB. Additional fusion protein variants can be producedby changing the composition or length of the linker peptide separatingthe antibody pharmaceutical from the HIRMAb.

In some embodiments, the anti-Aβ peptide antibody is specific forAβ¹⁻⁴⁰, Aβ¹⁻⁴², or Aβ¹⁻⁴³ peptide isoforms. Such isoform-specificanti-Aβ peptide antibodies are particularly useful where a ratio of Aβpeptide isoforms (e.g., Aβ¹⁻⁴²/Aβ¹⁻⁴⁰ ratio) is to be determined. Suchpeptide isoform ratio determinations are particularly useful forprognostic applications. For example, a high or increasing ratio ofAβ¹⁻⁴²/Aβ¹⁻⁴⁰ is indicative of Alzheimer's disease. See, e.g., Hanssonet al. (2007), Dement Geriatr Cogn Disord, 23(5):316-320.

In some embodiments, the anti-Aβ peptide antibody is a ScFv antibodycomprised of the variable region of the heavy chain (VH) and thevariable region of the light chain (VL) derived from the original murineanti-Aβ peptide antibody produced by a hybridoma. The amino acidsequence of the VH of the anti-Aβ peptide antibody is given in SEQ IDNO: 12. The amino acid sequence of the VL of the anti-Aβ peptideantibody is given in SEQ ID NO: 14. The amino acid sequence of the VH ofthe anti-Aβ peptide antibody joined to the VL anti-Aβ peptide antibodyvia a 17 amino acid linker, and containing the epitope of the 9E10antibody and a poly-histidine (H) tail at the carboxyl terminus, isgiven in SEQ ID NO: 16. The amino acid sequence of the VH of the anti-Aβpeptide antibody joined to the VL anti-Aβ peptide antibody via a 17amino acid linker, and containing the epitope of the 9E10 antibody atthe carboxyl terminus, and containing a 19 amino acid IgG signal peptideat the amino terminus, is given in SEQ ID NO: 18.

The VH of the anti-Aβ peptide ScFv antibody is comprised of 4 frameworkregions (FR), designated FR1, FR2, FR3, and FR4, and of 3complementarity determining regions (CDR), designated CDR1, CDR2, andCDR3. The VL of the anti-Aβ peptide ScFv antibody is comprised of 4 FRs,designated FR1, FR2, FR3, and FR4, and of 3 CDRs, designated CDR1, CDR2,and CDR3. The relationship of these 14 sub-domains are depicted in FIG.25. Amino acid substitutions in any of the 14 sub-domains could be madewith retention of the anti-Aβ peptide binding properties.

Accordingly, anti-Aβ peptide antibodies useful in the invention includeantibodies having at least about 60%, at least about 70%, at least about80%, at least about 90%, at least about 95%, at least about 99%, orgreater than 95% or greater than 99% sequence identity, e.g., 100%sequence identity, to the amino acid sequences disclosed herein.

Percent sequence identity is determined by conventional methods. See,for example, Altschul et al., Bull. Math. Bio. 48:603 (1986), andHenikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1992).Briefly, two amino acid sequences are aligned to optimize the alignmentscores using a gap opening penalty of 10, a gap extension penalty of 1,and the “BLOSUM62” scoring matrix of Henikoff and Henikoff (ibid.). Thepercent identity is then calculated as: ([Total number of identicalmatches]/[length of the longer sequence plus the number of gapsintroduced into the longer sequence in order to align the twosequences])(100).

Those skilled in the art appreciate that there are many establishedalgorithms available to align two amino acid sequences. The “FASTA”similarity search algorithm of Pearson and Lipman is a suitable proteinalignment method for examining the level of identity shared by an aminoacid sequence disclosed herein and the amino acid sequence of anotherpeptide. The FASTA algorithm is described by Pearson and Lipman, Proc.Nat'l Acad. Sci. USA 85:2444 (1988), and by Pearson, Meth. Enzymol.183:63 (1990). Briefly, FASTA first characterizes sequence similarity byidentifying regions shared by the query sequence (e.g., SEQ ID NO:21 orSEQ ID NO: 29) and a test sequence that have either the highest densityof identities (if the ktup variable is 1) or pairs of identities (ifktup=2), without considering conservative amino acid substitutions,insertions, or deletions. The ten regions with the highest density ofidentities are then rescored by comparing the similarity of all pairedamino acids using an amino acid substitution matrix, and the ends of theregions are “trimmed” to include only those residues that contribute tothe highest score. If there are several regions with scores greater thanthe “cutoff” value (calculated by a predetermined formula based upon thelength of the sequence and the ktup value), then the trimmed initialregions are examined to determine whether the regions can be joined toform an approximate alignment with gaps. Finally, the highest scoringregions of the two amino acid sequences are aligned using a modificationof the Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol.Biol. 48:444 (1970); Sellers, SIAM J. Appl. Math. 26:787 (1974)), whichallows for amino acid insertions and deletions. Illustrative parametersfor FASTA analysis are: ktup=1, gap opening penalty=10, gap extensionpenalty=1, and substitution matrix=BLOSUM62. These parameters can beintroduced into a FASTA program by modifying the scoring matrix file(“SMATRIX”), as explained in Appendix 2 of Pearson, Meth. Enzymol.183:63 (1990).

The present invention also includes peptides having a conservative aminoacid change, compared with an amino acid sequence disclosed herein. Manysuch changes have been described specifically. More generally, forexample, variants can be obtained that contain one or more amino acidsubstitutions of SEQ ID NO:22. In these variants, e.g., an alkyl aminoacid is substituted for an alkyl amino acid in either the VH or VL of ananti-Aβ peptide antibody amino acid sequence, an aromatic amino acid issubstituted for an aromatic amino acid in an anti-Aβ peptide antibodyamino acid sequence, a sulfur-containing amino acid is substituted for asulfur-containing amino acid in an anti-Aβ peptide antibody amino acidsequence, a hydroxy-containing amino acid is substituted for ahydroxy-containing amino acid in an anti-Aβ peptide antibody amino acidsequence, an acidic amino acid is substituted for an acidic amino acidin an anti-Aβ peptide antibody amino acid sequence, a basic amino acidis substituted for a basic amino acid in an anti-Aβ peptide antibodyamino acid sequence, or a dibasic monocarboxylic amino acid issubstituted for a dibasic monocarboxylic amino acid in an anti-Aβpeptide antibody amino acid sequence. Among the common amino acids, forexample, a “conservative amino acid substitution” is illustrated by asubstitution among amino acids within each of the following groups: (1)glycine, alanine, valine, leucine, and isoleucine, (2) phenylalanine,tyrosine, and tryptophan, (3) serine and threonine, (4) aspartate andglutamate, (5) glutamine and asparagine, and (6) lysine, arginine andhistidine. The BLOSUM62 table is an amino acid substitution matrixderived from about 2,000 local multiple alignments of protein sequencesegments, representing highly conserved regions of more than 500 groupsof related proteins (Henikoff and Henikoff, Proc. Nat'l Acad. Sci. USA89:10915 (1992)). Accordingly, the BLOSUM62 substitution frequencies canbe used to define conservative amino acid substitutions that may beintroduced into the amino acid sequences of the present invention.Although it is possible to design amino acid substitutions based solelyupon chemical properties (as discussed above), the language“conservative amino acid substitution” preferably refers to asubstitution represented by a BLOSUM62 value of greater than −1. Forexample, an amino acid substitution is conservative if the substitutionis characterized by a BLOSUM62 value of 0, 1, 2, or 3. According to thissystem, preferred conservative amino acid substitutions arecharacterized by a BLOSUM62 value of at least 1 (e.g., 1, 2 or 3), whilemore preferred conservative amino acid substitutions are characterizedby a BLOSUM62 value of at least 2 (e.g., 2 or 3). Further, in designingconservative amino acid substitutions, mutation tolerance predictionprograms can be used to greatly increase the number of functionalsequence variants so generated. Various programs for predicting theeffects of amino acid substitutions in a protein sequence on proteinfunction are described in, e.g., Henikoff et al. (2006), “Predicting theEffects of Amino Acid Substitutions on Protein Function,” Annu. Rev.Genomics Hum. Genet., 7:61-80. Such programs include, e.g., SIFT,PolyPhen, PANTHER PSEC, PMUT, and TopoSNP). These programs are availableto the public on the world wide web.

It also will be understood that amino acid sequences may includeadditional residues, such as additional N- or C-terminal amino acids,and yet still be essentially as set forth in one of the sequencesdisclosed herein, so long as the sequence retains sufficient biologicalprotein activity to be functional in the compositions and methods of theinvention.

Compositions of the invention are useful in one or more of transportingan agent, e.g., an antibody, across the BBB in the blood to braindirection; transporting an agent, e.g., antibody, across the BBB in thebrain to blood direction; and/or retaining activity of the agent, e.g.,antibody, once transported across the BBB.

Structures useful in transporting an agent across the BBB in the bloodto brain direction include structures capable of crossing theblood-brain barrier on an endogenous BBB receptor-mediated transporter,such as a transporter selected from the group consisting of the insulintransporter, the transferrin transporter, the leptin transporter, theLDL transporter, and the IGF receptor. In some embodiments, theendogenous BBB receptor-mediated transporter is selected from the groupconsisting of the insulin transporter and the transferrin transporter.In some embodiments, the endogenous BBB receptor-mediated transporter isthe insulin transporter, e.g., the human insulin transporter. Thus, inembodiments in which the composition is an antibody fusion protein, thepart of the antibody fusion protein that mediates transport across theBBB in the blood to brain direction is an immunoglobulin, and is anantibody to an endogenous BBB receptor-mediated transport system. Insome embodiments, the endogenous BBB receptor-mediated transport systemis selected from the group consisting of the BBB insulin receptor, theBBB transferrin receptor, the BBB leptin receptor, the BBB IGF receptor,or the BBB lipoprotein receptor. In some embodiments, the antibody is anantibody to the endogenous insulin BBB receptor-mediated transportsystem. Antibodies can be any suitable antibody as described herein. Thestructure capable of crossing the BBB can be an antibody, e.g., a MAbsuch as a chimeric MAb. The antibody can be an antibody to an endogenousBBB receptor-mediated transporter, as described herein.

In some embodiments, the invention provides compositions, e.g., a fusionprotein, that are capable of transport across the BBB from the CNS tothe blood, e.g., compositions that can cross the BBB in both directions.Thus, in some embodiments the compositions also include a structure thatis capable of crossing the BBB from the CNS to the blood. Any suitablestructure that is capable of crossing the BBB from the CNS to the bloodmay be used. In some embodiments, the invention utilizes structures thatare capable of crossing the BBB from the CNS to the blood via the Fcreceptor. The BBB expresses an Fc receptor (FcR), and the neonatal FcRor FcRn, and this FcR mediates the unidirectional efflux of IgGmolecules from brain to blood. See, e.g., Zhang, Y. and Pardridge, W. M.(2001): Mediated efflux of IgG molecules from brain to blood across theblood-brain barrier. J Neuroimmunol, 114: 168-172, and Schlachetzki, F.,Zhu, C. and Pardridge, W. M. (2002): Expression of the neonatal Fcreceptor (FcRn) at the blood-brain barrier. J Neurochem, 81: 203-206,incorporated herein by reference. The BBB FcR does not mediate theinflux of IgG molecules from blood to brain. The FcR binds the IgGmolecule at the interface of the CH2 and CH3 regions of the Fc part ofthe heavy chain. See, e.g., Martin, W. L., West, A. P., Jr., Gan, L. andBjorkman, P. J. (2001): Crystal structure at 2.8 A of anFcRn/heterodimeric Fc complex: mechanism of pH-dependent binding. MolCell, 7: 867-877, incorporated herein by reference. Thus, in someembodiments, the interface of the CH2 and CH3 regions of the Fc part ofthe heavy chain serve as a structure that can transport compositions ofthe invention from the CNS to the blood across the BBB. For example, theinterface of the CH2 and CH3 regions of the Fc part of the heavy chain,which is intact in the fusion antibody as illustrated in FIGS. 25 and26, is such a structure

It will be appreciated that the structure that crosses the BBB from theCNS to the blood, e.g., the interface of the CH2 and CH3 regions, may bepart of the structure that crosses the BBB from the blood to the CNS,e.g., an antibody directed to a receptor-mediated BBB transport system,e.g., the HIR system.

In embodiments used to treat aggregate diseases, were it not for theexport of the fusion antibody from brain back to blood via the FcR, thenthere would be little or no clearance of the monomeric proteins from theprotein aggregate. Many brain diseases are “aggregate” diseases, whichare caused by the gradual deposition of aggregated protein in the brain.The aggregates of AD are formed by the Aβ amyloid peptide; theaggregates of PD are formed by α-synuclein and/or parkin; the aggregatesof mad cow disease are formed by the Prp scrapie protein; the aggregatesof HD are formed by the huntingtin protein. Antibodies against themonomers of these aggregates can dissolve the aggregate, as illustratedfor Aβ aggregates in the Examples and in FIG. 41. Following binding ofthe aggregated protein by the fusion antibody, the complex of the fusionantibody and the aggregate precursor are exported out of brain and backto blood across the BBB, as shown in FIG. 27.

The Examples show that the fusion antibody binds the HIR with highaffinity, and that the fusion antibody crosses the BBB via thisreceptor. However, once the fusion antibody binds the aggregated proteinin brain, the fusion antibody must be able to efflux from brain back toblood, as depicted in FIG. 27.

In some embodiments, the structure that is capable of crossing the BBButilizes an endogenous BBB receptor mediated transport system, such as asystem that utilizes the insulin receptor, transferrin receptor, leptinreceptor, LDL receptor, or IGF receptor. In some embodiments, theendogenous BBB receptor mediated transport system is the insulin BBBreceptor mediated transport system. In some embodiments, the structurethat is capable of crossing the BBB is an antibody, e.g., a monoclonalantibody (MAb) such as a chimeric MAb. The antibody can be a chimericantibody with sufficient human sequence that it is suitable foradministration to a human. The antibody can be glycosylated ornonglycosylated; in some embodiments, the antibody is glycosylated,e.g., in a glycosylation pattern produced by its synthesis in a CHOcell. In embodiments in which the structure is an antibody, the covalentlinkage between the antibody and the neurotherapeutic agent may be alinkage between any suitable portion of the antibody and the antibodypharmaceutical agent, as long as it allows the antibody-agent fusion tocross the blood-brain barrier and the antibody pharmaceutical agent toretain a therapeutically useful portion of its activity within the CNS.In certain embodiments, the covalent link is between one or more lightchains of the targeting antibody and the antibody pharmaceutical agent.

In some embodiments, more than one type of structure capable of crossingthe BBB, e.g., molecular Trojan horse, may be used. The differentstructures may be covalently attached to a single antibodypharmaceutical agent, e.g., a single ScFv such as the anti-Aβ ScFv, ormultiple ScFv's, or any combination thereof. Thus, for example, in someembodiments either with the same ScFv attached to each MTH or adifferent ScFv attached, or combinations of ScFv attached. Thus theantibody pharmaceutical can be fused to multiple molecular Trojan horsesthat undergo receptor-mediated transport across the blood-brain barrier,including monoclonal antibodies to the insulin receptor, transferrinreceptor, insulin-like growth factor (IGF) receptor, or the low densitylipoprotein (LDL) receptor or the endogenous ligand, including insulin,transferrin, the IGFs, or LDL. Ligands that traverse the blood-brainbarrier via absorptive-mediated transport may also be used as molecularTrojan horses including cationic proteins, or carbohydrate bearingproteins that bind to membrane lectins. The molecular weight range ofmolecular Trojan horses is 1000 Daltons to 500,000 Daltons.

The covalent linkage between the structure capable of crossing the BBBand the antibody pharmaceutical agent may be direct (e.g., a peptidebond between the terminal amino acid of one peptide and the terminalamino acid of the other peptide to which it is linked) or indirect, viaa linker. If a linker is used, it may be any suitable linker, e.g., apeptide linker. If a peptide linker is used, it may be 1, 2, 3, 4, 5, 6,7, 8, 9, 10, or more than 10 amino acids in length. In some embodiments,a two amino acid linker is used. In some embodiments, the linker has thesequence ser-ser. The covalent linkage may be cleavable, however this isnot a requirement for activity of the system in some embodiments;indeed, an advantage of these embodiments of the present invention isthat the fusion protein, without cleavage, is partially or fully activeboth for transport and for activity once across the BBB.

In some embodiments, a noncovalent attachment may be used. An example ofnoncovalent attachment of the MTH, e.g., MAb, to the large moleculetherapeutic neuroprotective factor is avidin/streptavidin-biotinattachment. Such an approach is further described in U.S. Pat. No.6,287,792, entitled “Drug delivery of antisense oligodeoxynucleotidesand peptides to tissues in vivo and to cells using avidin-biotintechnology, which is hereby incorporated by reference in its entirety.

The agents transported across the BBB may be any suitable agent forwhich such transport is desired in one or both directions. For example,the agent may be a therapeutic, diagnostic, or research agent.Particularly useful agents for transport include antibodies, e.g.,antibody pharmaceuticals that are active in the CNS.

The antibody pharmaceutical that is active in the CNS can be aneurotherapeutic agent, e.g., an antibody that disaggregates insolubleprotein in the brain. In some embodiments, the antibody pharmaceuticalis directed against the Aβ amyloid peptide of Alzheimer's disease (AD)for the diagnosis or treatment of AD. In some embodiments, the antibodypharmaceutical is directed against α-synuclein of Parkinson's disease(PD) for the diagnosis or treatment of PD. In some embodiments, theantibody pharmaceutical is directed against the huntingtin protein ofHuntington's disease (HD) for the diagnosis or treatment of HD. In someembodiments, the antibody pharmaceutical is directed against the Prpprotein of scrapie or mad cow disease for the diagnosis or treatment ofhuman equivalents of scrapie. In some embodiments, the antibodypharmaceutical is directed against an envelope protein of the West Nilevirus for the diagnosis or treatment of West Nile encephalitis. In someembodiments, the antibody pharmaceutical is directed against the tumornecrosis factor (TNF) related apoptosis inducing ligand (TRAIL) for thediagnosis or treatment of acquired immune deficiency syndrome (AIDS),which infects the brain. In some embodiments, the antibodypharmaceutical is directed against the nogo A protein for the diagnosisor treatment of brain injury, spinal cord injury, or stroke. In someembodiments, the antibody pharmaceutical is directed against the HER2protein for the diagnosis or treatment of breast cancer metastatic tothe brain. In some embodiments, the antibody pharmaceutical is directedagainst an oncogenic receptor proteins such as the epidermal growthfactor receptor (EGFR) for the diagnosis or treatment of either primarybrain cancer or metastatic cancer of the brain. In some embodiments, theantibody pharmaceutical is directed against an oncogenic growth factorsuch as the epidermal growth factor (EGF) or the hepatocyte growthfactor (HGF) for the diagnosis or treatment of either primary braincancer or metastatic cancer of the brain. In some embodiments, theantibody pharmaceutical is directed against an oligodendrocyte surfaceantigen for the diagnosis or treatment of demyelinating disease such asmultiple sclerosis. The structure capable of crossing the BBB and theneurotherapeutic agent are covalently linked by a peptide linker in someembodiments.

Particularly useful antibody structures are ScFvs. In some embodiments,more than one molecule of the same therapeutic ScFv agent is attached tothe structure that crosses the BBB. For example, in compositions of theinvention where an ScFv is attached to an antibody, one molecule of theScFv is attached to each heavy chain, naturally producing a homodimerstructure. This is desired if a dimeric configuration of the ScFv isrequired for high antigen avidity. However, if a dimeric configurationis not required, then 2 different ScFv molecules with 2 differentantigen specificities could be fused to the heavy chain of the targetingantibody. A naturally occurring homo-dimeric structure between two ScFvmolecules is formed when the ScFv is fused to a carboxyl terminus of theCH3 region of an IgG molecule, as illustrated in FIG. 26. Without beingbound by theory, it is thought that this may account for the unexpectedfinding of essentially 100% of activity of binding of the fusionantibody to the Aβ amyloid peptide (see, e.g., FIG. 34).

In some embodiments, more than one type of ScFv agent can be attached tothe structure that is capable of crossing the blood-brain barrier. Insome embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 differentScFv agents may be attached to the structure that is capable of crossingthe blood-brain barrier. In certain embodiments, 2 different ScFv areattached to an antibody to an endogenous BBB receptor-mediated transportsystem. Any combination of ScFv may be used. Certain ScFv's may formhetero-dimeric structures, and in some embodiments the inventionprovides a fusion protein constructed of one ScFv monomer fused to onechain (e.g., heavy chain) of an antibody, e.g., of the HIRMAb, andanother ScFv monomer fused to the second chain of the antibody.Typically, the molecular weight range of recombinant ScFv's that may befused to the molecular Trojan horse ranges from 1000 Daltons to 500,000Daltons.

Compositions that Cross the BBB from Blood to CNS and from CNS to Blood.

In one aspect, the invention provides a composition that includes (i) afirst portion capable of crossing the BBB from the blood to the brainvia a first receptor-mediated BBB transport system; associated with (ii)a second portion capable of crossing the BBB from the brain to the bloodvia a second receptor-mediated BBB transport system. In someembodiments, the composition further contains (iii) a third portioncapable of interacting with a central nervous system component. Thecomposition can contain a protein, e.g., an antibody construct. In someembodiments, the first portion is capable of crossing the BBB on anendogenous BBB receptor mediated transport system that is the insulinreceptor, transferrin receptor, leptin receptor, lipoprotein receptor,or the IGF receptor. In some embodiments, the endogenous BBB receptormediated transport system is the insulin BBB receptor mediated transportsystem. In some embodiments, the second receptor-mediated BBB transportsystem includes the Fc receptor system. In some embodiments, the firstand second portions are part of an antibody structure, e.g., the secondportion comprises the CH2-CH3 region of the antibody structure. In someembodiments, the third portion comprises an antibody, antibody fragment,or ScFv. In embodiments containing a third portion capable ofinteracting with a central nervous system component, the central nervoussystem component can be a pathological substance associated with a braindisorder, e.g., Alzheimer's disease, Parkinson's disease, Huntington'sdisease, bovine spongiform encephalopathy, West Nile virus encephalitis,Neuro-AIDS, brain injury, spinal cord injury, metastatic cancer of thebrain, metastatic breast cancer of the brain, primary cancer of thebrain, or multiple sclerosis. The pathological substance may one or moreof a protein, nucleic acid, carbohydrate, carbohydrate polymer, lipid,glycolipid, small molecule, or combinations thereof. In someembodiments, the pathological substance is a protein, e.g., Aβ amyloid,α-synuclein, huntingtin Protein, PrP prion protein, West Nile envelopeprotein, tumor necrosis factor (TNF) related apoptosis inducing ligand(TRAIL), Nogo A, HER2, epidermal growth factor receptor (EGFR),hepatocyte growth factor (HGF), or oligodendrocyte surface antigen. Insome embodiments, the protein is Aβ amyloid

In some embodiments, the invention provides a composition that includes(i) a first portion capable of crossing the BBB from the blood to thebrain via a first receptor-mediated BBB transport system; associatedwith (ii) a second portion capable of crossing the BBB from the brain tothe blood via a second receptor-mediated BBB transport system; and (iii)a third portion capable of interacting with a central nervous systemcomponent, where the first portion and the second portions are part ofan antibody, and the third portion is a ScFv. The antibody can bedirected to an endogenous BBB receptor-mediated transport system, e.g.,the insulin BBB receptor mediated transport system such as the humaninsulin receptor mediated transport system. In some embodiments, theScFv is a ScFv to Aβ amyloid peptide of AD.

In some embodiments, the invention provides compositions containing afusion protein, where the targeting MAb is an antibody to the humaninsulin BBB receptor mediated transport system linked to an anti-AβScFv. The ScFv is linked via its amino terminus to the carboxy terminusof the heavy chain of the antibody by a ser-ser linker. The antibody canbe a chimeric antibody with sufficient human sequence that it issuitable for administration to a human. In some embodiments, theinvention provides compositions containing a fusion MAb with a heavychain-ScFv fusion protein and a separate covalently linked light chain,where the light chain is at least about 60%, or about 70%, or about 80%,or about 90%, or about 95%, or about 99% identical to, or issubstantially 100% identical to amino acids 21-234 of SEQ ID NO: 29, andthe heavy chain-ScFv fusion is at least about 60%, or about 70%, orabout 80%, or about 90%, or about 95%, or about 99% identical to, or issubstantially 100% identical to amino acids 20-708 of SEQ ID NO: 22.

The invention also provides compositions containing an antibodypharmaceutical that is covalently linked to a chimeric MAb to the humanBBB insulin receptor. In some embodiments, the heavy chain of the MAb iscovalently linked to the pharmaceutical antibody to form a fusionprotein. The antibody pharmaceutical can be any antibody pharmaceuticaldescribed herein, i.e., any antibody pharmaceutical for which transportacross the BBB is desired. In some embodiments, the antibodypharmaceutical is antibody against aggregated protein in brain, e.g., Aβamyloid as in AD.

Compositions that Cross the BBB in Both Directions and that are Capableof Interacting with a CNS Component.

In one aspect, the invention provides a composition containing (i) afirst portion capable of crossing the BBB from the blood to the brain;(ii) a second portion capable of interacting with a central nervoussystem component; and (iii) a third portion capable of crossing the BBBfrom the brain to the blood, where the first, second, and third portionsare linked and wherein the first, second, and third portions are not thesame and do not share common structures. In some embodiments, thecomposition is a non-naturally-occurring composition. In someembodiments, the first and third portions include a protein, e.g. anantibody, such as a mAb. In some embodiments, the first, second, andthird portions are covalently linked. In some embodiments, the firstportion is capable of crossing the BBB from the blood to the brain viaan endogenous BBB receptor mediated transport system, e.g., the insulinreceptor, transferrin receptor, leptin receptor, lipoprotein receptor,or the IGF receptor. In some embodiments, the endogenous BBB receptormediated transport system is the insulin BBB receptor mediated transportsystem. In some embodiments, the second portion includes an antibody,antibody fragment, or ScFv The central nervous system component withwhich the second portion interacts can include a pathological substanceassociated with a brain disorder, e.g., Alzheimer's disease, Parkinson'sdisease, Huntington's disease, bovine spongiform encephalopathy, WestNile virus encephalitis, Neuro-AIDS, brain injury, spinal cord injury,metastatic cancer of the brain, metastatic breast cancer of the brain,primary cancer of the brain, or multiple sclerosis. In some embodiments,the pathological substance is of a type selected from the groupconsisting of proteins, nucleic acids, carbohydrates, carbohydratepolymers, lipids, glycolipids, and small molecules, e.g. a protein suchas Aβ amyloid, α-synuclein, huntingtin Protein, PrP prion protein, WestNile envelope protein, tumor necrosis factor (TNF) related apoptosisinducing ligand (Nogo A, HER2, epidermal growth factor receptor (EGFR),hepatocyte growth factor (HGF), or oligodendrocyte surface antigen. Insome embodiments, the protein is Aβ amyloid. In some embodiments, thethird portion includes a structure that is capable of crossing the BBBfrom the brain to the blood via a receptor mediated BBB transport systemsuch as the Fc receptor system, e.g., a structure that is part of anantibody structure, such as the CH2-CH3 region of the antibodystructure.

Compositions that Contain a ScFv that is Bonded to an Immunoglobulin andRetains Activity/Affinity.

In another aspect, the invention provides a composition containing aScFv that binds an antigen, where (i) the ScFv is derived from a firstimmunoglobulin, (ii) the ScFv is bonded with a second immunoglobulin,wherein the second immunoglobulin is optionally an immunoglobulin thatis modified from its native form; and (iii) the affinity of the ScFv forits antigen is more than about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100,110, or 120% of the first immunoglobulin from which the ScFv wasderived. In some embodiments, the ScFv is covalently bonded to thesecond immunoglobulin, e.g., at its amino terminus to the secondimmunoglobulin, or at its carboxy terminus to the second immunoglobulin.The ScFv can be bonded to the carboxy terminus of the heavy chain or thelight chain of the second immunoglobulin, e.g., to the carboxy terminusof the heavy chain of the second immunoglobulin. The ScFv can also bebonded to the amino terminus of the heavy or light chain of the secondimmunoglobulin. In some embodiments, the ScFv is bonded to the CH3region of the heavy chain of the second immunoglobulin. In someembodiments, the second immunoglobulin has been modified so that itsheavy chain is truncated, and the ScFv is bonded to the carboxy terminusof the truncated heavy chain. In some of these embodiments, the ScFv isbonded to the carboxy terminus of the truncated heavy chain, and theheavy chain has been truncated so that its carboxy terminus lies withina region of the native heavy chain selected from the group consisting ofthe CH1, hinge, CH2, and CH3 regions. In some embodiments, the ScFv isbonded to the amino terminus of the heavy chain or the light chain ofthe second immunoglobulin. In some embodiments, the ScFv is derived froman antibody directed against a pathological substance present in thebrain, where the pathological substance is associated with a braindisorder, e.g., Alzheimer's disease, Parkinson's disease, Huntington'sdisease, bovine spongiform encephalopathy, West Nile virus encephalitis,Neuro-AIDS, brain injury, spinal cord injury, metastatic cancer of thebrain, metastatic breast cancer of the brain, primary cancer of thebrain, or multiple sclerosis. The pathological substance can of a typeselected from the group consisting of proteins, nucleic acids,carbohydrates, carbohydrate polymers, lipids, glycolipids, and smallmolecules, e.g., a protein such as Aβ amyloid, α-synuclein, huntingtinProtein, PrP prion protein, West Nile envelope protein, tumor necrosisfactor (TNF) related apoptosis inducing ligand (TRAIL), Nogo A, HER2,epidermal growth factor receptor (EGFR), hepatocyte growth factor (HGF),or oligodendrocyte surface antigen.

In the case of a ScFv antibody pharmaceutical agent (e.g., a ScFvagainst the Aβ peptide of AD), the ScFv can be covalently linked by itscarboxy or amino terminus to the carboxy or amino terminus of the lightchain (LC) or heavy chain (HC) of the targeting antibody. Any suitablelinkage may be used, e.g., carboxy terminus of light chain to aminoterminus of ScFv, carboxy terminus of heavy chain to amino terminus ofScFv, amino terminus of light chain to carboxy terminus of ScFv, aminoterminus of heavy chain to carboxy terminus of ScFv, carboxy terminus oflight chain to carboxy terminus of ScFv, carboxy terminus of heavy chainto carboxy terminus of ScFv, amino terminus of light chain to aminoterminus of ScFv, or amino terminus of heavy chain to amino terminus ofScFv. In some embodiments, the linkage is from the carboxy terminus ofthe HC to the amino terminus of the ScFv, where the VH precedes the VLof the ScFv. In other embodiments, the VL could precede the VH of theScFv. It will be appreciated that a linkage between terminal amino acidsis not required, and any linkage which meets the requirements of theinvention may be used; such linkages between non-terminal amino acids ofpeptides are readily accomplished by those of skill in the art.

In some embodiments, the invention utilizes a ScFv against the Aβamyloid peptide of AD. Strikingly, it has been found that fusionproteins of these forms of the ScFv retain normal or even greater thannormal transport and activity. It is surprising that the affinity of theantibody fusion protein for the Aβ amyloid peptide is the same as theaffinity of the original 150 kDa murine MAb against the Aβ amyloidpeptide, because the fusion protein is comprised of a ScFv derived fromthe original murine MAb against the Aβ amyloid peptide. Generally, theaffinity and/or avidity of a ScFv for the target antigen is reducedcompared to the original MAb. High avidity for the target antigen isderived from the bivalent interaction between the intact 150 kDa MAb andthe antigen. In contrast, the interaction of the ScFv and the antigen ismonovalent. In addition, it is generally recognized that when a ScFv isfused to another antibody, the affinity of the ScFv for the targetantigen is reduced. However, in the design of the fusion antibodydepicted in FIG. 26, the bivalent interaction between the antigen andthe ScFv is restored. The production of this new genetically engineeredantibody fusion protein creates a tri-functional molecule that (i) bindswith high affinity to the HIR to cause influx across the BBB from bloodto brain, (ii) binds with high affinity to the FcR to cause effluxacross the BBB from brain to blood, and (iii) binds with high affinityto the Aβ amyloid peptide of AD to cause disaggregation of amyloidplaque.

Strikingly, it has been found that multifunctional antibody fusionproteins of the invention, e.g., tri-functional fusion proteins, retaina high proportion of the activity of the separate portions, e.g., theportion that is capable of crossing the BBB and the portion that isactive in the CNS. Accordingly, the invention further provides a fusionprotein containing a structure capable of crossing the BBB in eitherdirection, covalently linked to an antibody pharmaceutical that isactive in the central nervous system (CNS), where the structure capableof crossing the blood-brain barrier and the antibody pharmaceutical thatis active in the central nervous system each retain an average of atleast about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, or 120% oftheir activities, compared to their activities as separate entities. Insome embodiments, the invention provides a fusion protein containing astructure capable of crossing the BBB, covalently linked to an antibodypharmaceutical that is active in the central nervous system (CNS), wherethe structure capable of crossing the blood-brain barrier and theantibody pharmaceutical that is active in the central nervous systemeach retain an average of at least about 50% of their activities,compared to their activities as separate entities. In some embodiments,the invention provides a fusion protein containing a structure capableof crossing the BBB, covalently linked to a antibody pharmaceutical thatis active in the central nervous system (CNS), where the structurecapable of crossing the blood-brain barrier and the antibodypharmaceutical that is active in the central nervous system each retainan average of at least about 60% of their activities, compared to theiractivities as separate entities. In some embodiments, the inventionprovides a fusion protein containing a structure capable of crossing theBBB, covalently linked to a antibody pharmaceutical that is active inthe central nervous system (CNS), where the structure capable ofcrossing the blood-brain barrier and the antibody pharmaceutical that isactive in the central nervous system each retain an average of at leastabout 70% of their activities, compared to their activities as separateentities. In some embodiments, the invention provides a fusion proteincontaining a structure capable of crossing the BBB, covalently linked toa antibody pharmaceutical that is active in the central nervous system(CNS), where the structure capable of crossing the blood-brain barrierand the antibody pharmaceutical that is active in the central nervoussystem each retain an average of at least about 80% of their activities,compared to their activities as separate entities. In some embodiments,the invention provides a fusion protein containing a structure capableof crossing the BBB, covalently linked to a antibody pharmaceutical thatis active in the central nervous system (CNS), where the structurecapable of crossing the blood-brain barrier and the antibodypharmaceutical that is active in the central nervous system each retainan average of at least about 90% of their activities, compared to theiractivities as separate entities. In some embodiments, the structurecapable of crossing the blood-brain barrier retains at least about 10,20, 30, 40, 50, 60, 70, 80, 90, 95, 99, or 100% of its activity,compared to its activity as a separate entity, and the antibodypharmaceutical that is active in the central nervous system retains atleast about 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99, or 100% of itsactivity, compared to its activity as a separate entity.

As used herein, “activity” includes physiological activity (e.g.,ability to cross the BBB and/or therapeutic activity), and also bindingaffinity of the structures for their respective receptors or targetantigens.

Transport of the structure capable of crossing the BBB across the BBBmay be compared for the structure alone and for the structure as part ofa fusion structure of the invention by standard methods. For example,pharmacokinetics and brain uptake of the fusion structure, e.g., fusionprotein, by a model animal, e.g., a mammal such as a primate, may beused. Such techniques are illustrated in Example 10, which demonstratesthe binding of the antibody fusion protein to the purified human insulinreceptor. Similarly, standard models for the function of the antibodypharmaceutical, e.g. the therapeutic or protective function of aantibody therapeutic agent, may also be used to compare the function ofthe agent alone and the function of the agent as part of a fusionstructure of the invention. See, e.g., Example 10, which demonstratesthe activity of an murine anti-Aβ MAb alone and a ScFv derived from thismurine anti-Aβ MAb, wherein the ScFv is bound to a fusion protein in amodel system (Aβ peptide binding). In Example 10, Example 11, andExample 12, the fusion protein of the invention retained about 50-100%of the transport ability and the therapeutic function of its individualcomponents, i.e., a structure capable of crossing the BBB (a MAb to thehuman insulin receptor) and an anti-Aβ ScFv antibody pharmaceutical.

Alternatively, functional assays may be used as a marker of activity.Transport of the fusion protein across the primate BBB in vivo iscompared to the chimeric HIRMAb in Example 11. The bloodpharmacokinetics, and the pattern of brain uptake of antibody fusionprotein and the chimeric HIRMAb are, on average, nearly identical.Binding of the fusion antibody to the amyloid in autopsy sections of ADbrain is compared for the murine anti-Aβ MAb and the fusion protein inExample 10, Example 13, and Example 14. The binding of either antibodyto the amyloid plaque of AD is comparable. “Average” measurements arethe average of at least three separate measurements.

Compositions for Transporting Antibodies from Brain to Blood

In another aspect, the invention provides a non-naturally-occurringcomposition comprising a portion that is capable of transporting anantibody structure from the brain to the blood across the BBB. In someembodiments, the transport is via the BBB FcR. In some embodiments, theantibody structure is a therapeutic or diagnostic antibody structure,such as a therapeutic or diagnostic antibody structure interacts with apathological substance, wherein the pathological substance is associatedwith a brain disorder, e.g., Alzheimer's disease, Parkinson's disease,Huntington's disease, bovine spongiform encephalopathy, West Nile virusencephalitis, Neuro-AIDS, brain injury, spinal cord injury, metastaticcancer of the brain, metastatic breast cancer of the brain, primarycancer of the brain, or multiple sclerosis. The pathological substancecan be one or more of proteins, nucleic acids, carbohydrates,carbohydrate polymers, lipids, glycolipids, small molecules, orcombinations thereof. In some embodiments, the pathological substance isa protein, e.g., Aβ amyloid, α-synuclein, huntingtin Protein, PrP prionprotein, West Nile envelope protein, tumor necrosis factor (TNF) relatedapoptosis inducing ligand (TRAIL), Nogo A, HER2, epidermal growth factorreceptor (EGFR), hepatocyte growth factor (HGF), or oligodendrocytesurface antigen. In some embodiments, the protein is Aβ amyloid. Theantibody structure can be a single chain Fv antibody (ScFv). Thetherapeutic antibody structure or diagnostic antibody structure can linkto a structure that is capable of crossing the blood-brain barrier(BBB). The portion that is capable of transporting an antibody structurefrom the brain to the blood across the BBB can interact with the Fcreceptor.

The composition can further contain a portion that is capable ofcrossing the BBB from the blood to the brain, such as a portion that iscapable of crossing the BBB crosses the BBB on an endogenous BBBreceptor mediated transport system, e.g., the insulin receptor,transferrin receptor, leptin receptor, lipoprotein receptor, or the IGFreceptor. In some embodiments, the endogenous BBB receptor mediatedtransport system is the insulin BBB receptor mediated transport system.

Compositions Containing ScFv Peptide Sequences.

In still another aspect, the invention provides a composition containinga ScFv, wherein the VH region of the ScFv comprises a sequence that isat least 80, 90, 95, or 99% identical to SEQ ID NO: 12. In someembodiments, the VL region of the ScFv contains a sequence that is atleast 80, 90, 95, or 99% identical to SEQ ID NO: 14. The ScFv can linkto an Ab, e.g., a MAb. Ab or MAb is directed to an endogenous BBBreceptor-mediated transport system, e.g., the insulin receptor,transferrin receptor, leptin receptor, lipoprotein receptor, or the IGFreceptor. In some embodiments, the endogenous BBB receptor mediatedtransport system is the insulin BBB receptor mediated transport system.The linkage may be covalent. In some embodiments, the ScFv is linked tothe carboxy terminus of the light chain of the Ab or MAb. In someembodiments, the ScFv is linked via its amino terminus to the carboxyterminus of the light chain of the Ab or MAb. In some embodiments, theScFv is linked via its carboxy terminus to the carboxy terminus of thelight chain of the Ab or MAb. In some embodiments, the ScFv is linked tothe carboxy terminus of the heavy chain of the Ab or MAb. In someembodiments, the ScFv is linked via its amino terminus to the carboxyterminus of the heavy chain of the Ab or MAb. In some embodiments, theScFv is linked via its carboxy terminus to the carboxy terminus of theheavy chain of the Ab or MAb.

In some embodiments, the invention provides a composition containing aScFv where the VH region of the ScFv contains (i) a CDR1 sequence thatis at least about 60, 70, 80, 90, 95, 99, or 100% identical to thesequence of amino acids 26-35 of SEQ ID NO: 12; (ii) a CDR2 sequencethat is at least about 60, 70, 80, 90, 95, 99, or 100% identical to thesequence of amino acids 50-66 of SEQ ID NO: 12; and (iii) a CDR3sequence that is at least about 60, 70, 80, 90, 95, 99, or 100%identical to the sequence of amino acids 99-103 of SEQ ID NO: 12.

In some embodiments, the invention provides a composition containing aScFv where the VL region of the ScFv contains (i) a CDR1 sequence thatis at least about 60, 70, 80, 90, 95, 99, or 100% identical to thesequence of amino acids 24-39 of SEQ ID NO: 14; (ii) a CDR2 sequencethat is at least about 60, 70, 80, 90, 95, 99, or 100% identical to thesequence of amino acids 55-61 of SEQ ID NO: 14; and (iii) a CDR3sequence that is at least about 60, 70, 80, 90, 95, 99, or 100%identical to the sequence of amino acids 94-102 of SEQ ID NO: 14.

Compositions Capable of Achieving Distribution of an Antibody in theBrain after Peripheral Administration.

In another aspect, the invention provides a composition containing atherapeutic antibody structure or diagnostic antibody structure, wherethe composition is capable of achieving an average volume ofdistribution in the brain of the neurotherapeutic antibody structure ordiagnostic antibody structure of at least about 20, 30, 40, 50, 60, 70,80, 80, 90, or 100 uL/gram brain following peripheral administration. Insome embodiments, the therapeutic antibody structure or diagnosticantibody structure is capable of binding to a pathological substancepresent in the brain, where the pathological substance is associatedwith a brain disorder such as Alzheimer's disease, Parkinson's disease,Huntington's disease, bovine spongiform encephalopathy, West Nile virusencephalitis, Neuro-AIDS, brain injury, spinal cord injury, metastaticcancer of the brain, metastatic breast cancer of the brain, primarycancer of the brain, or multiple sclerosis. In some embodiments, thepathological substance is a protein, nucleic acid, carbohydrate,carbohydrate polymer, lipid, glycolipid, small molecule, or combinationthereof. In some embodiments, the pathological substance is a protein,e.g. Aβ amyloid, α-synuclein, huntingtin Protein, PrP prion protein,West Nile envelope protein, tumor necrosis factor (TNF) relatedapoptosis inducing ligand (TRAIL), Nogo A, HER2, epidermal growth factorreceptor (EGFR), hepatocyte growth factor (HGF), or oligodendrocytesurface antigen. In some embodiments, the protein is Aβ amyloid. In someembodiments, the therapeutic antibody structure is a single chain Fvantibody (ScFv). In some embodiments, the therapeutic antibody structureor diagnostic antibody structure is linked (e.g., covalently linked) toa structure that is capable of crossing the blood-brain barrier (BBB),such as a structure that is capable of crossing the BBB crosses the BBBon an endogenous BBB receptor mediated transport system, e.g., theinsulin receptor, transferrin receptor, leptin receptor, lipoproteinreceptor, or the IGF receptor. In some embodiments, the endogenous BBBreceptor mediated transport system is the insulin BBB receptor mediatedtransport system. In some embodiments, the structure that is capable ofcrossing the BBB is capable of crossing the BBB from blood to brain andfrom brain to blood. In some of these embodiments, the structure that iscapable of crossing the BBB is capable of crossing the BBB from blood tobrain via a first receptor-mediated transport system and from brain toblood via a second receptor-mediated transport system. The firstreceptor-mediated transport system can be, e.g., the insulin receptor,transferrin receptor, leptin receptor, lipoprotein receptor, or the IGFreceptor. In some embodiments, the first receptor mediated transportsystem is the insulin BBB receptor mediated transport system. In someembodiments, the second receptor-mediated transport system is theFc-receptor-mediated transport system. The structure that is capable ofcrossing the BBB can be an antibody, e.g., a mAb as described herein.

Accordingly, in some embodiments, the invention provides compositionscontaining an antibody pharmaceutical agent covalently linked to astructure that is capable of crossing the blood-brain barrier (BBB),where the composition is capable of producing an average increase inbrain volume of distribution of the antibody pharmaceutical of more thanabout 20, 30, 40, 50, 60, 70, 80, 80, 90, or 100 uL/gram brain followingperipheral administration. The invention also provides compositionscontaining an antibody pharmaceutical that is covalently linked to achimeric MAb to the human BBB insulin receptor. The invention furtherprovides a fusion protein containing a structure capable of crossing theBBB, covalently linked to an antibody pharmaceutical that is active inthe central nervous system (CNS), where the structure capable ofcrossing the blood-brain barrier and the antibody pharmaceutical that isactive in the central nervous system each retain an average of at leastabout 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99, 100, 110, or 120% oftheir activities, compared to their activities as separate entities. Theinvention also provides pharmaceutical compositions that contain one ormore compositions of the invention and a pharmaceutically acceptableexcipient.

In some embodiments, the invention provides compositions containing anantibody pharmaceutical agent covalently linked to a structure that iscapable of crossing the blood-brain barrier (BBB), where the compositionis capable of producing an average elevation of volume of distributionin the brain of the antibody pharmaceutical agent of at least about 20,30, 40, 50, 60, 70, 80, 80, 90, or 100 uL/gram brain followingperipheral administration.

“Elevation” of the agent is an increase in the brain volume ofdistribution of the pharmaceutical antibody compared to theconcentration of the pharmaceutical antibody administered alone (i.e.,not covalently linked to a structure that is capable of crossing theBBB) in different individuals. The individual in which the elevation ismeasured is a mammal, such as a rat, or, preferably, a primate, e.g., amonkey. An example of measurements of elevation of the level of apharmaceutical antibody is given in FIG. 38.

The antibody pharmaceutical agent may be any suitable antibody agent, asdescribed herein. In some embodiments, the antibody pharmaceutical isdirected against the Aβ amyloid peptide of Alzheimer's disease (AD) forthe diagnosis or treatment of AD. In some embodiments, the antibodypharmaceutical is directed against α-synuclein of Parkinson's disease(PD) for the diagnosis or treatment of PD. In some embodiments, theantibody pharmaceutical is directed against the huntingtin protein ofHuntington's disease (HD) for the diagnosis or treatment of HD. In someembodiments, the antibody pharmaceutical is directed against the Prpprotein of scrapie or mad cow disease for the diagnosis or treatment ofhuman equivalents of scrapie. In some embodiments, the antibodypharmaceutical is directed against an envelope protein of the West Nilevirus for the diagnosis or treatment of West Nile encephalitis. In someembodiments, the antibody pharmaceutical is directed against the tumornecrosis factor (TNF) related apoptosis inducing ligand (TRAIL) for thediagnosis or treatment of acquired immune deficiency syndrome (AIDS),which infects the brain. In some embodiments, the antibodypharmaceutical is directed against the nogo A protein for the diagnosisor treatment of brain injury, spinal cord injury, or stroke. In someembodiments, the antibody pharmaceutical is directed against the HER2protein for the diagnosis or treatment of breast cancer metastatic tothe brain. In some embodiments, the antibody pharmaceutical is directedagainst an oncogenic receptor proteins such as the epidermal growthfactor receptor (EGFR) for the diagnosis or treatment of either primarybrain cancer or metastatic cancer of the brain. In some embodiments, theantibody pharmaceutical is directed against an oncogenic growth factorsuch as the epidermal growth factor (EGF) or the hepatocyte growthfactor (HGF) for the diagnosis or treatment of either primary braincancer or metastatic cancer of the brain. In some embodiments, theantibody pharmaceutical is directed against an oligodendrocyte surfaceantigen for the diagnosis or treatment of demyelinating disease such asmultiple sclerosis. In some embodiments, the antibody pharmaceutical isan ScFv against the Aβ peptide of AD, and contains a sequence that is atleast about 60, 70, 80, 90, 95, 99, or 100% identical to the sequence ofamino acids 465-708 of SEQ ID NO: 22. In some embodiments, the antibodypharmaceutical is an ScFv against the Aβ peptide of AD, and contains aVH domain with sequence that is at least about 60, 70, 80, 90, 95, 99,or 100% identical to the sequence of amino acids of SEQ ID NO: 12. Insome embodiments, the antibody pharmaceutical is an ScFv against the Aβpeptide of AD, and contains a VL domain with sequence that is at leastabout 60, 70, 80, 90, 95, 99, or 100% identical to the sequence of aminoacids of SEQ ID NO: 14. In some embodiments, the antibody pharmaceuticalis an ScFv against the Aβ peptide of AD, and contains a VH domain withCDR1, CDR2, CDR3 sequences that are at least about 60, 70, 80, 90, 95,99, or 100% identical to the sequence of amino acids 26-35, 50-66, and99-103 of SEQ ID NO: 12, respectively. In some embodiments, the antibodypharmaceutical is an ScFv against the Aβ peptide of AD, and contains aVL domain with CDR1, CDR2, CDR3 sequences that are at least about 60,70, 80, 90, 95, 99, or 100% identical to the sequence of amino acids24-39, 55-61, and 94-102 of SEQ ID NO: 14, respectively. In someembodiments, the antibody pharmaceutical is an ScFv against the Aβpeptide of AD, and contains a VH domain with FR1, FR2, FR3, FR4sequences that are at least about 60, 70, 80, 90, 95, 99, or 100%identical to the sequence of amino acids 1-25, 36-49, 67-98, 104-114 ofSEQ ID NO: 12, respectively. In some embodiments, the antibodypharmaceutical is an ScFv against the Aβ peptide of AD, and contains aVL domain with FR1, FR2, FR3, FR4 sequences that are at least about 60,70, 80, 90, 95, 99, or 100% identical to the sequence of amino acids1-23, 40-54, 62-93, 103-113 of SEQ ID NO: 14, respectively.

In some embodiments, the invention provides compositions containing anantibody pharmaceutical agent covalently linked to a structure that iscapable of crossing the BBB where the composition is capable ofproducing an average increase in brain volume of distribution of theantibody pharmaceutical of 20, 30, 40, 50, 60, 70, 80, 80, 90, or 100uL/gram brain following peripheral administration, where the antibodypharmaceutical agent is a ScFv against aggregated protein and thestructure that is capable of crossing the BBB is a targeting MAb to anendogenous BBB receptor mediated transport system. The targetingantibody can be glycosylated or nonglycosylated; in some embodiments,the antibody is glycosylated, e.g., in a glycosylation pattern producedby its synthesis in a CHO cell. In certain embodiments, the antibodypharmaceutical is an anti-Aβ ScFv. The targeting MAb can be an antibodyto the insulin BBB receptor mediated transport system, e.g., a chimericMAb. The targeting antibody can be a chimeric antibody with sufficienthuman sequence that it is suitable for administration to a human. Insome embodiments, the insulin receptor is a human insulin receptor andthe antibody pharmaceutical is a ScFv. In some embodiments, the ScFvcontains a sequence that is at least about 60, 70, 80, 90, 95, 99, or100% identical to the sequence of amino acids 1-244 of SEQ ID NO: 16.The ScFv can be covalently linked at its amino terminus to the carboxyterminus of the heavy chain of the targeting MAb, optionally with alinker between the termini, such as the two amino-acid linker ser-ser.In some embodiments, the heavy chain of the targeting MAb contains asequence that is at least about 60, 70, 80, 90, 95, 99, or 100%identical to amino acids 20-462 of SEQ ID NO: 28. In some embodiments,the heavy chain of the targeting MAb contains a VH domain with CDR1,CDR2, CDR3 sequences that are at least about 60, 70, 80, 90, 95, 99, or100% identical to the sequence of amino acids 45-54, 69-85, and 118-121of SEQ ID NO: 28, respectively. In some embodiments, the heavy chain ofthe targeting MAb contains a VH domain with FR1, FR2, FR3, FR4 sequencesthat are at least about 60, 70, 80, 90, 95, 99, or 100% identical to thesequence of amino acids 20-44, 55-68, 86-117, 122-132 of SEQ ID NO: 28,respectively. In some embodiments, the light chain of the targeting MAbcontains a sequence that is at least about 60, 70, 80, 90, 95, 99, or100% identical to amino acids 21-234 of SEQ ID NO: 29. In someembodiments, the light chain of the targeting MAb contains a VL domainwith CDR1, CDR2, CDR3 sequences that are at least about 60, 70, 80, 90,95, 99, or 100% identical to the sequence of amino acids 44-54, 70-76,and 109-117 of SEQ ID NO: 29, respectively. In some embodiments, thelight chain of the targeting MAb contains a VL domain with FR1, FR2,FR3, FR4 sequences that are at least about 60, 70, 80, 90, 95, 99, or100% identical to the sequence of amino acids 21-43, 55-69, 77-108,118-128 of SEQ ID NO: 29, respectively.

Pharmaceutical Compositions

The invention also provides pharmaceutical compositions that contain oneor more compositions of the invention and a pharmaceutically acceptableexcipient. A thorough discussion of pharmaceutically acceptablecarriers/excipients can be found in Remington's Pharmaceutical Sciences,Gennaro, A R, ed., 20th edition, 2000: Williams and Wilkins PA, USA.Pharmaceutical compositions of the invention include compositionssuitable for administration via any peripheral route, includingintravenous, subcutaneous, intramuscular, intraperitoneal injection;oral, rectal, transbuccal, pulmonary, transdermal, intranasal, or anyother suitable route of peripheral administration.

The compositions of the invention are particular suited for injection,e.g., as a pharmaceutical composition for intravenous, subcutaneous,intramuscular, or intraperitoneal administration. Aqueous compositionsof the present invention comprise an effective amount of a compositionof the present invention, which may be dissolved or dispersed in apharmaceutically acceptable carrier or aqueous medium. The phrases“pharmaceutically or pharmacologically acceptable” refer to molecularentities and compositions that do not produce an adverse, allergic orother untoward reaction when administered to an animal, e.g., a human,as appropriate. As used herein, “pharmaceutically acceptable carrier”includes any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents and thelike. The use of such media and agents for pharmaceutically activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active ingredient, its use inthe therapeutic compositions is contemplated. Supplementary activeingredients can also be incorporated into the compositions.

Exemplary pharmaceutically acceptable carriers for injectablecompositions can include salts, for example, mineral acid salts such ashydrochlorides, hydrobromides, phosphates, sulfates, and the like; andthe salts of organic acids such as acetates, propionates, malonates,benzoates, and the like. For example, compositions of the invention maybe provided in liquid form, and formulated in saline based aqueoussolution of varying pH (5-8), with or without detergents suchpolysorbate-80 at 0.01-1%, or carbohydrate additives, such mannitol,sorbitol, or trehalose. Commonly used buffers include histidine,acetate, phosphate, or citrate. Under ordinary conditions of storage anduse, these preparations can contain a preservative to prevent the growthof microorganisms. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol; phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate, and gelatin.

For human administration, preparations meet sterility, pyrogenicity,general safety, and purity standards as required by FDA and otherregulatory agency standards. The active compounds will generally beformulated for parenteral administration, e.g., formulated for injectionvia the intravenous, intramuscular, subcutaneous, intralesional, orintraperitoneal routes. The preparation of an aqueous composition thatcontains an active component or ingredient will be known to those ofskill in the art in light of the present disclosure. Typically, suchcompositions can be prepared as injectables, either as liquid solutionsor suspensions; solid forms suitable for use in preparing solutions orsuspensions upon the addition of a liquid prior to injection can also beprepared; and the preparations can also be emulsified.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, methods ofpreparation include vacuum-drying and freeze-drying techniques whichyield a powder of the active ingredient plus any additional desiredingredient from a previously sterile-filtered solution thereof.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. The formulations are easily administered in a variety ofdosage forms, such as the type of injectable solutions described above,but drug release capsules and the like can also be employed

The term “unit dose” refers to physically discrete units suitable foruse in a subject, each unit containing a predetermined-quantity of thetherapeutic composition calculated to produce the desired responses,discussed above, in association with its administration, i.e., theappropriate route and treatment regimen. The quantity to beadministered, both according to number of treatments and unit dose,depends on the subject to be treated, the state of the subject and theprotection desired. The person responsible for administration will, inany event, determine the appropriate dose for the individual subject.

The active therapeutic agents may be formulated within a mixture tocomprise about 0.0001 to 1.0 milligrams, or about 0.001 to 0.1milligrams, or about 1.0 to 100 milligrams or even about 0.01 to 1.0grams per dose or so. Multiple doses can also be administered. In someembodiments, a dosage of about 5 to about 50 mg of a fusion protein ofthe invention is used as a unit dose for administration to a human,e.g., about 5 to about 50 mg of a fusion protein of Aβ ScFv and a HIRMAb.

In addition to the compounds formulated for parenteral administration,such as intravenous or intramuscular injection, other alternativemethods of administration of the present invention may also be used,including but not limited to intradermal administration (See U.S. Pat.Nos. 5,997,501; 5,848,991; and 5,527,288), pulmonary administration (SeeU.S. Pat. Nos. 6,361,760; 6,060,069; and 6,041,775), buccaladministration (See U.S. Pat. Nos. 6,375,975; and 6,284,262),transdermal administration (See U.S. Pat. Nos. 6,348,210; and 6,322,808)and transmucosal administration (See U.S. Pat. No. 5,656,284). All suchmethods of administration are well known in the art. One may also useintranasal administration of the present invention, such as with nasalsolutions or sprays, aerosols or inhalants. Nasal solutions are usuallyaqueous solutions designed to be administered to the nasal passages indrops or sprays. Nasal solutions are prepared so that they are similarin many respects to nasal secretions. Thus, the aqueous nasal solutionsusually are isotonic and slightly buffered to maintain a pH of 5.5 to6.5. In addition, antimicrobial preservatives, similar to those used inophthalmic preparations and appropriate drug stabilizers, if required,may be included in the formulation. Various commercial nasalpreparations are known and include, for example, antibiotics andantihistamines and are used for asthma prophylaxis.

Additional formulations, which are suitable for other modes ofadministration, include suppositories and pessaries. A rectal pessary orsuppository may also be used. Suppositories are solid dosage forms ofvarious weights and shapes, usually medicated, for insertion into therectum or the urethra. After insertion, suppositories soften, melt ordissolve in the cavity fluids. For suppositories, traditional bindersand carriers generally include, for example, polyalkylene glycols ortriglycerides; such suppositories may be formed from mixtures containingthe active ingredient in any suitable range, e.g., in the range of 0.5%to 10%, preferably 1%-2%.

Oral formulations include such normally employed excipients as, forexample, pharmaceutical grades of mannitol, lactose, starch, magnesiumstearate, sodium saccharine, cellulose, magnesium carbonate and thelike. These compositions take the form of solutions, suspensions,tablets, pills, capsules, sustained release formulations, or powders. Incertain defined embodiments, oral pharmaceutical compositions willcomprise an inert diluent or assimilable edible carrier, or they may beenclosed in a hard or soft shell gelatin capsule, or they may becompressed into tablets, or they may be incorporated directly with thefood of the diet. For oral therapeutic administration, the activecompounds may be incorporated with excipients and used in the form ofingestible tablets, buccal tables, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. Such compositions andpreparations can contain at least 0.1% of active compound. Thepercentage of the compositions and preparations may, of course, bevaried, and may conveniently be between about 2 to about 75% of theweight of the unit, or between about 25-60%. The amount of activecompounds in such therapeutically useful compositions is such that asuitable dosage will be obtained.

The tablets, troches, pills, capsules and the like may also contain thefollowing: a binder, such as gum tragacanth, acacia, cornstarch, orgelatin; excipients, such as dicalcium phosphate; a disintegratingagent, such as corn starch, potato starch, alginic acid and the like; alubricant, such as magnesium stearate; and a sweetening agent, such assucrose, lactose or saccharin may be added or a flavoring agent, such aspeppermint, oil of wintergreen, or cherry flavoring. When the dosageunit form is a capsule, it may contain, in addition to materials of theabove type, a liquid carrier. Various other materials may be present ascoatings or to otherwise modify the physical form of the dosage unit.For instance, tablets, pills, or capsules may be coated with shellac,sugar or both. A syrup of elixir may contain the active compoundssucrose as a sweetening agent, methylene and propyl parabens aspreservatives, a dye and flavoring, such as cherry or orange flavor. Insome embodiments, an oral pharmaceutical composition may be entericallycoated to protect the active ingredients from the environment of thestomach; enteric coating methods and formulations are well-known in theart.

In some embodiments, the invention provides nucleic acids that code forproteins or peptides of the invention. In certain embodiments, theinvention provides a single nucleic acid sequence containing a firstsequence coding for a some or all of a light chain of a firstimmunoglobulin operably linked to a second sequence coding for some orall of a heavy chain of the first immunoglobulin, where either the firstsequence further codes for a ScFv derived from a second immunoglobulinthat is expressed as a fusion protein of the ScFv covalently linked tothe light chain or the second sequence further codes for a ScFv derivedfrom a second immunoglobulin that is expressed as a fusion protein ofthe ScFv covalently linked to the heavy chain. The first immunoglobulincan be directed to an endogenous BBB receptor mediated transport system,e.g., the insulin receptor, transferrin receptor, leptin receptor,lipoprotein receptor, or the IGF receptor. In some embodiments, theendogenous BBB receptor mediated transport system is the insulin BBBreceptor mediated transport system. The ScFv can be directed to apathological substance present in the brain, where the pathologicalsubstance is associated with a brain disorder such as Alzheimer'sdisease, Parkinson's disease, Huntington's disease, bovine spongiformencephalopathy, West Nile virus encephalitis, Neuro-AIDS, brain injury,spinal cord injury, metastatic cancer of the brain, metastatic breastcancer of the brain, primary cancer of the brain, or multiple sclerosis.The pathological substance is can be protein, nucleic acid,carbohydrate, carbohydrate polymer, lipid, glycolipid, small molecule,or a combination thereof. In some embodiments, the pathologicalsubstance is a protein, e.g., Aβ amyloid, α-synuclein, huntingtinProtein, PrP prion protein, West Nile envelope protein, tumor necrosisfactor (TNF) related apoptosis inducing ligand (TRAIL), Nogo A, HER2,epidermal growth factor receptor (EGFR), hepatocyte growth factor (HGF),or oligodendrocyte surface antigen. In some embodiments, thepathological protein is Aβ amyloid. In some embodiments, the VH regionof the ScFv contains a comprises a sequence that is at least about 80,90, 95, or 99% identical to SEQ ID NO: 12. In some embodiments, the VLregion of the ScFv contains a sequence that is at least 80, 90, 95, or99% identical to SEQ ID NO: 14.

The inventor further provides a vector containing a single nucleic acidsequence containing a first sequence coding for a some or all of a lightchain of a first immunoglobulin operably linked to a second sequencecoding for some or all of a heavy chain of the first immunoglobulin,where either the first sequence further codes for a ScFv derived from asecond immunoglobulin that is expressed as a fusion protein of the ScFvcovalently linked to the light chain or the second sequence furthercodes for a ScFv derived from a second immunoglobulin that is expressedas a fusion protein of the ScFv covalently linked to the heavy chain.

The invention further provides a cell containing a vector containing asingle nucleic acid sequence containing a first sequence coding for asome or all of a light chain of a first immunoglobulin operably linkedto a second sequence coding for some or all of a heavy chain of thefirst immunoglobulin, where either the first sequence further codes fora ScFv derived from a second immunoglobulin that is expressed as afusion protein of the ScFv covalently linked to the light chain or thesecond sequence further codes for a ScFv derived from a secondimmunoglobulin that is expressed as a fusion protein of the ScFvcovalently linked to the heavy chain. In some embodiments the cell is aeukaryotic cell. In some embodiments, the cell is a Chinese hamsterovary cell.

In some embodiments, the invention provides nucleic acid sequences thatare at least about 60, 70, 80, 90, 95, 99, or 100% identical to aparticular nucleotide sequence. For example, in some embodiments theinvention provides a single nucleic acid sequence containing a firstsequence coding for a some or all of a light chain of a firstimmunoglobulin operably linked to a second sequence coding for some orall of a heavy chain of the first immunoglobulin, where either the firstsequence further codes for a ScFv derived from a second immunoglobulinthat is expressed as a fusion protein of the ScFv covalently linked tothe light chain or the second sequence further codes for a ScFv derivedfrom a second immunoglobulin that is expressed as a fusion protein ofthe ScFv covalently linked to the heavy chain, where the VH region ofthe ScFv contains at least one, two, or three of: (i) a CDR1 sequencethat is at least about 60, 70, 80, 90, 95, 99, or 100% identical to thesequence of amino acids 26-35 of SEQ ID NO: 12; (ii) a CDR2 sequencethat is at least about 60, 70, 80, 90, 95, 99, or 100% identical to thesequence of amino acids 50-66 of SEQ ID NO: 12; and (iii) a CDR3sequence that is at least about 60, 70, 80, 90, 95, 99, or 100%identical to the sequence of amino acids 99-103 of SEQ ID NO: 12.

In some embodiments the invention provides a single nucleic acidsequence containing a first sequence coding for a some or all of a lightchain of a first immunoglobulin operably linked to a second sequencecoding for some or all of a heavy chain of the first immunoglobulin,where either the first sequence further codes for a ScFv derived from asecond immunoglobulin that is expressed as a fusion protein of the ScFvcovalently linked to the light chain or the second sequence furthercodes for a ScFv derived from a second immunoglobulin that is expressedas a fusion protein of the ScFv covalently linked to the heavy chain,where the VL region of the ScFv contains at least one, two, or three of:

(i) a CDR1 sequence that is at least about 60, 70, 80, 90, 95, 99, or100% identical to the sequence of amino acids 24-39 of SEQ ID NO: 14;(ii) a CDR2 sequence that is at least about 60, 70, 80, 90, 95, 99, or100% identical to the sequence of amino acids 55-61 of SEQ ID NO: 14;and (iii) a CDR3 sequence that is at least about 60, 70, 80, 90, 95, 99,or 100% identical to the sequence of amino acids 94-102 of SEQ ID NO:14.

In some embodiments, the invention provides a nucleic acid containing afirst sequence that is at least about 60, 70, 80, 90, 95, 99, or 100%identical to nucleotides 58-2127 of SEQ ID NO: 19 and a second sequencethat is at least about 60, 70, 80, 90, 95, 99, or 100% identical tonucleotides 801-1442 of SEQ ID NO: 27.

For sequence comparison, of two nucleic acids, typically one sequenceacts as a reference sequence, to which test sequences are compared. Whenusing a sequence comparison algorithm, test and reference sequences areentered into a computer, subsequence coordinates are designated, ifnecessary, and sequence algorithm program parameters are designated.Default program parameters can be used, or alternative parameters can bedesignated. The sequence comparison algorithm then calculates thepercent sequence identities for the test sequences relative to thereference sequence, based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well-known in the art. Optimal alignment of sequencesfor comparison can be conducted, including but not limited to, by thelocal homology algorithm of Smith and Waterman (1970) Adv. Appl. Math.2:482c, by the homology alignment algorithm of Needleman and Wunsch(1970) J. Mol. Biol. 48:443, by the search for similarity method ofPearson and Lipman (1988) Proc. Nat'l. Acad. Sci. USA 85:2444, bycomputerized implementations of these algorithms (GAP, BESTFIT, FASTA,and TFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.), or by manual alignment andvisual inspection (see, e.g., Ausubel et al., Current Protocols inMolecular Biology (1995 supplement)).

One example of an algorithm that is suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al. (1977) Nuc. AcidsRes. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410,respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information. TheBLAST algorithm parameters W, T, and X determine the sensitivity andspeed of the alignment. The BLASTN program (for nucleotide sequences)uses as defaults a wordlength (W) of 11, an expectation (E) or 10, M=5,N=−4 and a comparison of both strands. The BLAST algorithm is typicallyperformed with the “low complexity” filter turned off. The BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad.Sci. USA 90:5873-5787). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a nucleic acidis considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.2, more preferably less than about0.01, and most preferably less than about 0.001.

The invention provides nucleic acids that code for any of the peptidesof the invention. In some embodiments, the invention provides a singlenucleic acid sequence containing a gene coding for a light chain of atargeting immunoglobulin and a gene coding for a fusion protein, wherethe fusion protein includes a heavy chain of the targetingimmunoglobulin covalently linked to an antibody pharmaceutical. In someembodiments, the peptide is a therapeutic peptide. In some embodimentsthe antibody pharmaceutical is directed against aggregated protein suchas Aβ. In some embodiments, the anti-Aβ antibody pharmaceutical is aScFv. In some embodiments, the targeting immunoglobulin is an IgG. Insome embodiments, the targeting IgG is a MAb, such as a chimeric MAb.The targeting antibody can be an antibody to a transport system, e.g.,an endogenous BBB receptor-mediated transport system such as theendogenous BBB receptor-mediated insulin transport system. In someembodiments, the endogenous BBB receptor-mediated insulin transportsystem is a human endogenous BBB receptor-mediated insulin transportsystem and wherein the pharmaceutical to which the immunoglobulin heavychain is covalently linked is an anti-Aβ ScFv. Any suitable peptide,neurotherapeutic peptide, ScFv, antibody, monoclonal antibody, orchimeric antibody, as described herein, may be coded for by the nucleicacid, combined as a fusion protein and coded for in a single nucleicacid sequence. As is well-known in the art, owing to the degeneracy ofthe genetic code, any combination of suitable codons may be used to codefor the desired fusion protein. In addition, other elements useful inrecombinant technology, such as promoters, termination signals, and thelike, may also be included in the nucleic acid sequence. Such elementsare well-known in the art. In addition, all nucleic acid sequencesdescribed and claimed herein include the complement of the sequence.

In some embodiments that code for an anti-Aβ ScFv, as a component of thefusion protein, the ScFv contains a sequence that is about 60, 70, 80,90, 95, 99, or 100% identical to the sequence of amino acids 20-263 ofSEQ ID NO: 18. In some embodiment, the ScFv contains a sequence of theVH part that is about 60, 70, 80, 90, 95, 99, or 100% identical to thesequence of amino acids 1-114 of SEQ ID NO: 12. In some embodiment, theScFv contains a sequence of the VL part that is about 60, 70, 80, 90,95, 99, or 100% identical to the sequence of amino acids 1-113 of SEQ IDNO: 14. In some embodiment, the ScFv contains a sequence of the linkerpeptide joining the VH and the VL part that is about 60, 70, 80, 90, 95,99, or 100% identical to the sequence of amino acids 115-131 of SEQ IDNO: 16. In some embodiments, the ScFv is linked at its amino terminus tocarboxy terminus of the heavy chain of the targeting immunoglobulin,e.g., MAb. The heavy chain of the targeting MAb can comprise a sequencethat is about 60, 70, 80, 90, 95, 99 or 100% identical to amino acids20-462 of SEQ ID NO: 28. In some embodiments, the light chain of thetargeting immunoglobulin, e.g., MAb, comprises a sequence that is about60, 70, 80, 90, 95, 99 or 100% identical to amino acids 21-234 of SEQ IDNO: 29. The nucleic acid can further contain a nucleic acid sequencethat codes for a peptide linker between the heavy chain of the MAb andthe therapeutic antibody. In some embodiments, the linker is S-S. Thenucleic acid may further contain a nucleic acid sequence coding for asignal peptide, wherein the signal peptide is linked to the heavy chain.Any suitable signal peptide, as known in the art or subsequentlydeveloped, may be used. In some embodiments, the signal peptide attachedto the heavy chain comprises a sequence that is about 60, 70, 80, 90,95, 99, or 100% identical to amino acids 1-19 of SEQ ID NO: 28. In someembodiments, the nucleic acid contains a nucleic acid sequence codingfor another signal peptide, wherein the other signal peptide is linkedto the light chain. The signal peptide linked to the light chain cancomprise a sequence that is about 60, 70, 80, 90, 95, 99, or 100%identical to amino acids 1-20 of SEQ ID NO: 29. The nucleic acid cancontain a nucleic acid sequence coding for a selectable marker. In someembodiments the selectable marker is DHFR. The sequence of the DHFR canbe about 60, 70, 80, 90, 95, 99, or 100% identical to amino acids 1-187of SEQ ID NO: 30.

In certain embodiments, the invention provides a nucleic acid comprisinga first sequence that codes for an antibody pharmaceutical, e.g., a ScFvagainst Aβ, in the same open reading frame as a second sequence thatcodes for a targeting immunoglobulin component. The targetingimmunoglobulin component can be, e.g., a light chain or a heavy chain,e.g., that is at least about 60, 70, 80, 90, 95, 99, or 100% identicalto nucleotides 801-1442 of SEQ ID NO: 27 and a second sequence that isat least about 60, 70, 80, 90, 95, 99, or 100% identical to nucleotides2540-3868 of SEQ ID NO: 27. In some embodiments, the nucleic acid alsocontains a third sequence that is at least about 60, 70, 80, 90, 95, 99,or 100% identical to nucleotides 3874-4606 of SEQ ID NO: 27. In someembodiments, the nucleic acid further contains a fourth sequence thatcodes for a first signal peptide and a fifth sequence that codes for asecond signal peptide. In some embodiments, the fourth sequence is atleast about 60, 70, 80, 90, 95, 99, or 100% identical to nucleotides741-800 of SEQ ID NO: 27 and the fifth sequence is at least about 60,70, 80, 90, 95, 99, or 100% identical to nucleotides 2438-2539 of SEQ IDNO: 27. In some embodiments, the nucleic acid further contains asequence that codes for a selectable marker, such as dihydrofolatereductase (DHFR). In some embodiments, the sequence that codes for theDHFR is at least about 60, 70, 80, 90, 95, 99, or 100% identical tonucleotides 5618-5728 of SEQ ID NO: 27.

The invention also provides vectors. The vector can contain any of thenucleic acid sequences described herein. In some embodiments, theinvention provides a single tandem expression vector containing nucleicacid coding for an antibody heavy chain fused to an antibodypharmaceutical, e.g., a ScFv, and nucleic acid coding for a light chainof the antibody, all incorporated into a single piece of nucleic acid,e.g., a single piece of DNA. The single tandem vector can also includeone or more selection and/or amplification genes. A method of making anexemplary vector of the invention is provided in the Examples, and inFIG. 24. However, any suitable techniques, as known in the art, may beused to construct the vector.

The use of a single tandem vector has several advantages over previoustechniques. The transfection of a eukaryotic cell line withimmunoglobulin G (IgG) genes generally involves the co-transfection ofthe cell line with separate plasmids encoding the heavy chain (HC) andthe light chain (LC) comprising the IgG. In the case of an IgG fusionprotein, the gene encoding the recombinant therapeutic protein may befused to either the HC or LC gene. However, this co-transfectionapproach makes it difficult to select a cell line that has equally highintegration of both the HC and LC-fusion genes, or the HC-fusion and LCgenes. The approach to manufacturing the fusion protein utilized incertain embodiments of the invention is the production of a cell linethat is permanently transfected with a single plasmid DNA that containsall the required genes on a single strand of DNA, including theHC-fusion protein gene, the LC gene, the selection gene, e.g. neo, andthe amplification gene, e.g. the dihydrofolate reductase gene. As shownin the diagram of the fusion protein tandem vector in FIG. 24, theHC-fusion gene, the LC gene, the neo gene, and the DHFR gene are allunder the control of separate, but tandem promoters and separate buttandem transcription termination sequences. Therefore, all genes areequally integrated into the host cell genome, including the fusion geneof the therapeutic protein and either the HC or LC IgG gene.

Thus, in some embodiments the invention provides a vector containing asingle nucleic acid sequence containing a first sequence coding for asome or all of a light chain of a first immunoglobulin operably linkedto a second sequence coding for some or all of a heavy chain of thefirst immunoglobulin, where either the first sequence further codes fora ScFv derived from a second immunoglobulin that is expressed as afusion protein of the ScFv covalently linked to the light chain or thesecond sequence further codes for a ScFv derived from a secondimmunoglobulin that is expressed as a fusion protein of the ScFvcovalently linked to the heavy chain.

The invention further provides cells that incorporate one or more of thevectors of the invention. The cell may be a prokaryotic cell or aeukaryotic cell. In some embodiments, the cell is a eukaryotic cell. Insome embodiments, the cell is a mouse myeloma hybridoma cell. In someembodiments, the cell is a Chinese hamster ovary (CHO) cell. Exemplarymethods for incorporation of the vector(s) into the cell are given inthe Examples. However, any suitable techniques, as known in the art, maybe used to incorporate the vector(s) into the cell. In some embodiments,the invention provides a cell capable of expressing an immunoglobulinfusion protein, where the cell is a cell into which has been permanentlyintroduced a single tandem expression vector, where both theimmunoglobulin light chain gene and the gene for the immunoglobulinheavy chain fused to the antibody pharmaceutical, are incorporated intoa single piece of nucleic acid, e.g., DNA. In some embodiments, theinvention provides a cell capable of expressing an immunoglobulin fusionprotein, where the cell is a cell into which has been permanentlyintroduced a single tandem expression vector, where both theimmunoglobulin heavy chain gene and the gene for the immunoglobulinlight chain fused to the antibody pharmaceutical, are incorporated intoa single piece of nucleic acid, e.g., DNA. The introduction of thetandem vector may be by, e.g., permanent integration into the chromsomalnucleic acid, or by, e.g., introduction of an episomal genetic element.

Thus, in some embodiments the invention further provides a cellcontaining a vector containing a single nucleic acid sequence containinga first sequence coding for a some or all of a light chain of a firstimmunoglobulin operably linked to a second sequence coding for some orall of a heavy chain of the first immunoglobulin, where either the firstsequence further codes for a ScFv derived from a second immunoglobulinthat is expressed as a fusion protein of the ScFv covalently linked tothe light chain or the second sequence further codes for a ScFv derivedfrom a second immunoglobulin that is expressed as a fusion protein ofthe ScFv covalently linked to the heavy chain. In some embodiments thecell is a eukaryotic cell. In some embodiments, the cell is a Chinesehamster ovary cell.

In addition, the invention provides methods of manufacture. In someembodiments, the invention provides a method of manufacturing animmunoglobulin fusion protein, where the fusion protein contains animmunoglobulin heavy chain fused to a antibody pharmaceutical, bypermanently introducing into a eukaryotic cell a single tandemexpression vector, where both the immunoglobulin light chain gene andthe gene for the immunoglobulin heavy chain fused to the antibodypharmaceutical, are incorporated into a single piece of nucleic acid,e.g., DNA. In some embodiments, the invention provides a method ofmanufacturing an immunoglobulin fusion protein, where the fusion proteincontains an immunoglobulin light chain fused to a antibodypharmaceutical, by permanently introducing into a eukaryotic cell asingle tandem expression vector, where both the immunoglobulin heavychain gene and the gene for the immunoglobulin light chain fused to theantibody pharmaceutical, are incorporated into a single piece of nucleicacid, e.g., DNA. In some embodiments, the introduction of the vector isaccomplished by permanent integration into the host cell genome. In someembodiments, the introduction of the vector is accomplished byintroduction of an episomal genetic element containing the vector intothe host cell. Episomal genetic elements are well-known in the art Insome embodiments, the therapeutic agent is a antibody pharmaceutical. Insome embodiments, the single piece of nucleic acid further includes oneor more genes for selectable markers. In some embodiments, the singlepiece of nucleic acid further includes one or more amplification genes.In some embodiments, the immunoglobulin is an IgG, e.g., a MAb such as achimeric MAb. The methods may further include expressing theimmunoglobulin fusion protein, and/or purifying the immunoglobulinfusion protein. Exemplary methods for manufacture, including expressionand purification, are given in the Examples.

However, any suitable techniques, as known in the art, may be used tomanufacture, optionally express, and purify the proteins. These includenon-recombinant techniques of protein synthesis, such as solid phasesynthesis, manual or automated, as first developed by Merrifield anddescribed by Stewart et al. in Solid Phase Peptide Synthesis (1984).Chemical synthesis joins the amino acids in the predetermined sequencestarting at the C-terminus. Basic solid phase methods require couplingthe C-terminal protected α-amino acid to a suitable insoluble resinsupport. Amino acids for synthesis require protection on the α-aminogroup to ensure proper peptide bond formation with the preceding residue(or resin support). Following completion of the condensation reaction atthe carboxyl end, the α-amino protecting group is removed to allow theaddition of the next residue. Several classes of α-protecting groupshave been described, see Stewart et al. in Solid Phase Peptide Synthesis(1984), with the acid labile, urethane-based tertiary-butyloxycarbonyl(Boc) being the historically preferred. Other protecting groups, and therelated chemical strategies, may be used, including the base labile9-fluorenylmethyloxycarbonyl (FMOC). Also, the reactive amino acidsidechain functional groups require blocking until the synthesis iscompleted. The complex array of functional blocking groups, along withstrategies and limitations to their use, have been reviewed by Bodanskyin Peptide Synthesis (1976) and, Stewart et al. in Solid Phase PeptideSynthesis (1984).

Solid phase synthesis is initiated by the coupling of the describedC-terminal α-protected amino acid residue. Coupling requires activatingagents, such as dicyclohexycarbodiimide with or without1-hydroxybenzo-triazole, diisopropylcarbodiimide, orethyldimethylaminopropylcarbodiimide. After coupling the C-terminalresidue, the α-amino protected group is removed by trifluoroacetic acid(25% or greater) in dichloromethane in the case of acid labiletertiary-butyloxycarbonyl (Boc) groups. A neutralizing step withtriethylamine (10%) in dichloro-methane recovers the free amine (versusthe salt). After the C-terminal residue is added to the resin, the cycleof deprotection, neutralization and coupling, with intermediate washsteps, is repeated in order to extend the protected peptide chain. Eachprotected amino acid is introduced in excess (three to five fold) withequimolar amounts of coupling reagent in suitable solvent. Finally,after the completely blocked peptide is assembled on the resin support,reagents are applied to cleave the peptide form the resin and to removethe side chain blocking groups. Anhydrous hydrogen fluoride cleaves theacid labile tertiary-butyloxycarbonyl (Boc) chemistry groups. Severalnucleophilic scavengers, such as dimethylsulfide and anisole, areincluded to avoid side reactions especially on side chain functionalgroups.

Thus, in some embodiments, the invention provides a method ofmanufacturing an immunoglobulin fusion protein, where the fusion proteincomprises an immunoglobulin heavy chain fused to an antibody structureor an immunoglobulin light chain fused to an antibody structure, bypermanently introducing (e.g., integrating) into a eukaryotic cell asingle tandem expression vector, where the gene for the fusion proteinand another gene comprising the gene for the immunoglobulin light chainor the gene for the immunoglobulin heavy chain, are incorporated into asingle piece of DNA. The fusion protein can contain an immunoglobulinheavy chain fused to an antibody structure, where both the gene for thefusion protein and the gene for the immunoglobulin light chain areincorporated into a single piece of DNA. The fusion protein can containan immunoglobulin light chain fused to a therapeutic agent where boththe gene for the fusion protein and the gene for the immunoglobulinheavy chain are incorporated into a single piece of DNA. In someembodiments, the permanently introducing is achieved by introducing areplicating episomal genetic element containing the tandem vector intothe eukaryotic cell. In some embodiments, the antibody structure is aScFv. The method may further include incorporating one or more genes forselectable markers in said single piece of DNA. The method may furtherinclude incorporating one or more amplification genes in said singlepiece of DNA. The immunoglobulin can be an IgG. The immunoglobulin canbe a MAb. In some embodiments, the ScFv is directed against apathological substance associated with a brain disorder. In someembodiments, the pathological substance is of a type selected from thegroup consisting of proteins, nucleic acids, carbohydrates, carbohydratepolymers, lipids, glycolipids, and small molecules. In some embodiments,the pathological substance is a protein. The method can further includeexpressing the immunoglobulin fusion protein. The method can furtherinclude purifying the immunoglobulin fusion protein.

The invention also provides methods. In some embodiments, the inventionprovides methods for transport of an antibody pharmaceutical active inthe CNS across the BBB in an effective amount. In some embodiments, theinvention provides diagnostic, therapeutic, or research methods.

Diagnostic Methods

The invention provides diagnostic, prognostic, and treatment evaluationmethods. In some embodiments, the invention provides a method ofdiagnosis, prognosis, or treatment evaluation by measurement ofperipheral blood markers. In some embodiments, the invention provides amethod of diagnosis, prognosis, or treatment evaluation by imaging ofCNS structures associated with disease.

In some embodiments, the invention provides method of diagnosis,prognosis, or evaluation of treatment of a brain disorder by detecting asignal emitted by a composition in the CNS of an individual, where thecomposition includes an antibody that is capable of crossing the BBBfrom the blood to the brain and interacting with a pathologicalsubstance associated with a brain disorder. In some embodiments, themethod further includes administering the composition to the individual.The composition may be constructed so as to emit a signal, e.g., to emitpositrons, to give a radioactive signal, or to give a magnetic signal.In some embodiments, the composition is a radiopharmaceutical or amagnetopharmaceutical. In some embodiments, the composition is labeledwith a substance that emits the signal. In some embodiments, thesubstance that emits the signal is selected from the group consisting ofpositron emitters, radionuclide, and magnetic substances. In someembodiments, the substance that emits the signal is a positron emitter.In some embodiments, the substance that emits the signal is aradionuclide. In some embodiments, the brain disorder is selected fromthe group consisting of Alzheimer's disease, Parkinson's disease,Huntington's disease, bovine spongiform encephalopathy, West Nile virusencephalitis, Neuro-AIDS, brain injury, spinal cord injury, metastaticcancer of the brain, metastatic breast cancer of the brain, primarycancer of the brain, and multiple sclerosis. In some embodiments, thebrain disorder is Alzheimer's disease. The pathological substance can beof a type selected from the group consisting of proteins, nucleic acids,carbohydrates, carbohydrate polymers, lipids, glycolipids, and smallmolecules. In some embodiments, the pathological substance is a protein,e.g., Aβ amyloid, α-synuclein, huntingtin Protein, PrP prion protein,West Nile envelope protein, tumor necrosis factor (TNF) relatedapoptosis inducing ligand (TRAIL), Nogo A, HER2, epidermal growth factorreceptor (EGFR), hepatocyte growth factor (HGF), or oligodendrocytesurface antigen. In some embodiments, the protein is Aβ amyloid. In someembodiments, the antibody comprises a ScFv.

Thus, diagnostic, prognostic, and treatment evaluation methods includethe use of an antibody, e.g., positron emitter labeled, radiolabeled ormagnetically labeled antibodies capable of transport across the BBB,such as the fusion of a diagnostic antibody to a targeting agent such asan MAb for an endogenous receptor in the BBB, followed by the positron,magnetic or radiolabelling of the fusion protein, followed by systemicadministration, and external imaging of the localization within thebrain of the antibody diagnostic. The fusion antibody can be labeledwith a positron emitter for brain scanning using positron emissiontomography (PET), or labeled with a radionuclide that could be detectedwith single photon emission computed tomography (SPECT), or magneticallylabeled for MRI. For SPECT scanning, the fusion protein can beradiolabeled with 111-indium following conjugation to the fusionantibody of a suitable chelating agent. One such chelating agent is1,4,7,10-tetraazacyclododecane-N,—N′,N″,N′″-tetraacetic acid (DOTA).Administration is as described herein, and imaging may be achieved bymethods well known in the art.

In some embodiments, the invention provides a method of diagnosis,prognosis, or evaluation of treatment of a CNS disorder by measuring thelevel of a composition in a body fluid of an individual, where thecomposition is capable of crossing the BBB from the blood to the brain,interacting with a pathological substance associated with a braindisorder, and crossing the BBB from the brain to the blood, and wherethe composition has been administered to the individual and hasinteracted with the pathological substance. In some embodiments, thebrain disorder is Alzheimer's disease, Parkinson's disease, Huntington'sdisease, bovine spongiform encephalopathy, West Nile virus encephalitis,Neuro-AIDS, brain injury, spinal cord injury, metastatic cancer of thebrain, metastatic breast cancer of the brain, primary cancer of thebrain, or multiple sclerosis. In some embodiments, the brain disorder isAlzheimer's disease. In some embodiments, the pathological substance isof a type selected from the group consisting of proteins, nucleic acids,carbohydrates, carbohydrate polymers, lipids, glycolipids, smallmolecules, or combinations thereof. In some embodiments, thepathological substance is a protein, e.g., Aβ amyloid, α-synuclein,huntingtin Protein, PrP prion protein, West Nile envelope protein, tumornecrosis factor (TNF) related apoptosis inducing ligand (TRAIL), Nogo A,HER2, epidermal growth factor receptor (EGFR), hepatocyte growth factor(HGF), oroligodendrocyte surface antigen. In some embodiments, theprotein is Aβ amyloid. The method may further include administering thecomposition to the individual. The composition may include an antibody,and may also include a ScFv. The body fluid in some embodiments isblood, serum, or plasma. Methods of measuring a marker in a body fluidare well-known, e.g., sandwich based ELISA may be used.

Therapeutic Methods

The invention provides methods of treatment of CNS disorders orconditions by peripheral administration of an agent that does notnormally cross the BBB, e.g., an antibody, in a composition that iscapable of crossing the BBB from the blood to the brain. In someembodiments, the methods further include transport of the agent, e.g.,the antibody (typically bound to antigen) from the brain to the blood.For treatment of aggregation diseases, the latter step can be importantin allowing the disaggregated protein exit from the brain or CNS,without which the protein may reaggregate or cause other harm.

The compositions of the invention are effective in therapeutic methodsof the invention, and any suitable composition described herein may beused in the methods.

Thus, in some embodiments, the invention provides a method of treating aCNS disorder by administering to an individual suffering from thedisorder an effective amount of a composition containing a firststructure capable of crossing the BBB from the blood to the brain, asecond structure capable of interacting with a pathological substanceassociated with the disorder, and, optionally, a third structure capableof crossing the BBB from the brain to the blood. In some embodiments,the first and third structures (if a third structure is present)comprise an antibody, e.g., an antibody to an endogenous BBB receptormediated transport system, as described herein. In some embodiments, thesecond structure comprises a ScFv, as described herein. The ScFv may bedirected against a pathological substance associated with the disorder.In some embodiments, the pathological substance is of a type selectedfrom the group consisting of proteins, nucleic acids, carbohydrates,carbohydrate polymers, lipids, glycolipids, and small molecules. In someembodiments, the pathological substance is a protein, e.g., Aβ amyloid,α-synuclein, huntingtin Protein, PrP prion protein, West Nile envelopeprotein, tumor necrosis factor (TNF) related apoptosis inducing ligand(TRAIL), Nogo A, HER2, epidermal growth factor receptor (EGFR),hepatocyte growth factor (HGF), or oligodendrocyte surface antigen. Insome embodiments, the protein is Aβ amyloid. The method of administeringcan be any suitable method that introduces the agent into the peripheralcirculation, e.g., oral, intravenous, intramuscular, subcutaneous,intraperitoneal, rectal, transbuccal, intranasal, transdermal, orinhalation. In some embodiments, the administering is intravenous,intramuscular, or subcutaneous. In some embodiments, the CNS disorder isan aggregate CNS disorder. In some embodiments, the CNS disorder isAlzheimer's disease, Parkinson's disease, Huntington's disease, bovinespongiform encephalopathy West Nile virus encephalitis, Neuro-AIDS,brain injury, spinal cord injury, metastatic cancer of the brain,metastatic breast cancer of the brain, primary cancer of the brain, ormultiple sclerosis. In some embodiments, the CNS disorder is Alzheimer'sdisease. The individual can be an animal, e.g., a mammal. In someembodiments, the individual is a human. In some embodiments, theindividual is administered a dose of the composition that is about 1 toabout 100 mg.

In some embodiments of the invention, the methods involve administrationof a composition that includes an antibody structure that is useful intherapy or diagnosis of the disorder of interest. Monoclonal antibodydrug development illustrates the problems encountered when developmentof the delivery of agents active in the CNS, e.g., CNS drug development,is undertaken in the absence of a parallel program in delivery acrossthe BBB, e.g., CNS drug delivery. The advances in the molecularneurosciences during the Decade of the Brain of the 1990s, andsubsequently, led to identification of multiple targets in the brain formonoclonal antibody-based pharmaceuticals, including an antibodypharmaceutical directed against the Aβ amyloid peptide of Alzheimer'sdisease (AD) for the diagnosis or treatment of AD; an antibodypharmaceutical directed against α-synuclein of Parkinson's disease (PD)for the diagnosis or treatment of PD; an antibody pharmaceuticaldirected against the huntingtin protein of Huntington's disease (HD) forthe diagnosis or treatment of HD; an antibody pharmaceutical directedagainst the Pip protein of scrapie or mad cow disease for the diagnosisor treatment of human equivalents of scrapie; an antibody pharmaceuticaldirected against an envelope protein of the West Nile virus for thediagnosis or treatment of West Nile encephalitis; an antibodypharmaceutical directed against the tumor necrosis factor (TNF) relatedapoptosis inducing ligand (TRAIL) for the diagnosis or treatment ofacquired immune deficiency syndrome (AIDS), which infects the brain; anantibody pharmaceutical directed against the nogo A protein for thediagnosis or treatment of brain injury, spinal cord injury, or stroke;an antibody pharmaceutical directed against the HER2 protein for thediagnosis or treatment of breast cancer metastatic to the brain; anantibody pharmaceutical directed against an oncogenic receptor proteinssuch as the epidermal growth factor receptor (EGFR) for the diagnosis ortreatment of either primary brain cancer or metastatic cancer of thebrain; an antibody pharmaceutical directed against an oncogenic growthfactor such as the epidermal growth factor (EGF) or the hepatocytegrowth factor (HGF) for the diagnosis or treatment of either primarybrain cancer or metastatic cancer of the brain; or an antibodypharmaceutical directed against an oligodendrocyte surface antigen forthe diagnosis or treatment of demyelinating disease such as multiplesclerosis. In none of these cases, can the antibody pharmaceutical bedeveloped as a neuropharmaceutical for human disease, because theantibodies do not cross the BBB.

Owing to the BBB problem, antibody therapeutics must be injecteddirectly into the brain to achieve a therapeutic effect. It is notexpected that antibody pharmaceuticals will have beneficial effects onbrain disorders following the peripheral (intravenous, subcutaneous)administration of these molecules, because the molecules do not crossthe BBB.

Antibody pharmaceuticals can be developed as drugs for the brain thatare administered by peripheral routes of administration, providing theantibody is enabled to cross the BBB. Attachment of the antibodypharmaceutical, e.g. an anti-Aβ ScFv to a MTH, e.g., the chimericHIRMAb, offers a new approach to the non-invasive delivery of antibodytherapeutics to the CNS in animals, e.g., mammals such as humans for thetreatment of acute brain and spinal cord conditions, such as focal brainischemia, global brain ischemia, and spinal cord injury, and chronictreatment of neurodegenerative disease, including prion diseases,Alzheimer's disease, Parkinson's disease, Huntington's disease, ormultiple sclerosis, for the treatment of brain infection, such asinfection by the West Nile virus or the human immunodeficiency virus,and for the treatment of brain cancer, such as metastatic cancer tobrain, or primary brain cancer.

Accordingly, in some embodiments the invention provides methods oftransport of an antibody pharmaceutical active in the CNS from theperipheral circulation across the BBB in an effective amount, where theagent is covalently attached to a structure that crosses the BBB, andwhere the antibody pharmaceutical alone is not transported across theBBB in an effective amount. In some embodiments the invention providesmethods of transport of antibody pharmaceuticals from the peripheralcirculation across the BBB in a therapeutically effective amount, wherethe antibody pharmaceutical is covalently attached to a structure thatcrosses the BBB, and where the antibody pharmaceutical alone is nottransported across the BBB in a therapeutically effective amount.

The invention also provides, in some embodiments, methods of treatmentof disorders of the CNS by peripheral administration of an effectiveamount of a antibody pharmaceutical, e.g., an anti-aggregate antibodycovalently linked to a structure that is capable of crossing the BBB,where the antibody pharmaceutical alone is not capable of crossing theBBB in an effective amount when administered peripherally. In someembodiments, the CNS disorder is an acute disorder, and, in some cases,may require only a single administration of the agent. In someembodiments, the CNS disorder is a chronic disorder and requires morethan one administration of the agent.

In some embodiments, the effective amount, e.g., therapeuticallyeffective amount is such that a concentration in the brain is reached ofat least about 0.001, 0.01, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,20, 25, 30, 35, 40, 45, 50, 100, or more than 100 ng/gram brain. In someembodiments, a therapeutically effective amount, e.g., of a antibodypharmaceutical, is such that a brain level is achieved of about 0.1 to1000, or about 1-100, or about 5-50 ng/g brain. In some embodiments, theantibody pharmaceutical is directed against the Aβ amyloid peptide ofAlzheimer's disease (AD) for the diagnosis or treatment of AD. In someembodiments, the antibody pharmaceutical is directed against α-synucleinof Parkinson's disease (PD) for the diagnosis or treatment of PD. Insome embodiments, the antibody pharmaceutical is directed against thehuntingtin protein of Huntington's disease (HD) for the diagnosis ortreatment of HD. In some embodiments, the antibody pharmaceutical isdirected against the Prp protein of scrapie or mad cow disease for thediagnosis or treatment of human equivalents of scrapie. In someembodiments, the antibody pharmaceutical is directed against an envelopeprotein of the West Nile virus for the diagnosis or treatment of WestNile encephalitis. In some embodiments, the antibody pharmaceutical isdirected against the tumor necrosis factor (TNF) related apoptosisinducing ligand (TRAIL) for the diagnosis or treatment of acquiredimmune deficiency syndrome (AIDS), which infects the brain. In someembodiments, the antibody pharmaceutical is directed against the nogo Aprotein for the diagnosis or treatment of brain injury, spinal cordinjury, or stroke. In some embodiments, the antibody pharmaceutical isdirected against the HER2 protein for the diagnosis or treatment ofbreast cancer metastatic to the brain. In some embodiments, the antibodypharmaceutical is directed against an oncogenic receptor proteins suchas the epidermal growth factor receptor (EGFR) for the diagnosis ortreatment of either primary brain cancer or metastatic cancer of thebrain. In some embodiments, the antibody pharmaceutical is directedagainst an oncogenic growth factor such as the epidermal growth factor(EGF) or the hepatocyte growth factor (HGF) for the diagnosis ortreatment of either primary brain cancer or metastatic cancer of thebrain. In some embodiments, the antibody pharmaceutical is directedagainst an oligodendrocyte surface antigen for the diagnosis ortreatment of demyelinating disease such as multiple sclerosis.

In some embodiments, the invention provides methods of treating adisorder of the CNS by peripherally administering to an individual inneed of such treatment an effective amount of a antibody pharmaceutical,where the antibody pharmaceutical is capable of crossing the BBB toproduce an average elevation of antibody pharmaceutical concentration inthe brain of at least about 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20,25, 30, 35, 40, 45, 50, 100, or more than 100 ng/gram brain followingsaid peripheral administration, and where the antibody pharmaceuticalremains at the elevated level for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,or more than 10 days after a single administration. In some embodiments,the antibody pharmaceutical remains at a level of greater than about 1ng/g brain, or about 2 ng/g brain, or about 5 ng/g brain for about 2, 3,4, 5, 6, 7, 8, 9, 10, or more than 10 days after a singleadministration. In some embodiments, the antibody pharmaceutical is ananti-Aβ ScFv.

In some embodiments, the invention provides methods of treating adisorder of the CNS by peripherally administering to an individual inneed of such treatment an effective amount of a composition of theinvention. The term “peripheral administration,” as used herein,includes any method of administration that is not direct administrationinto the CNS, i.e., that does not involve physical penetration ordisruption of the BBB. “Peripheral administration” includes, but is notlimited to, intravenous intramuscular, subcutaneous, intraperitoneal,intranasal, transbuccal, transdermal, rectal, transalveolar(inhalation), or oral administration. Any suitable composition of theinvention, as described herein, may be used. In some embodiments, thecomposition is a antibody pharmaceutical covalently linked to a chimericHIR-MAb. In some embodiments, the antibody pharmaceutical is an anti-AβScFv.

A “disorder of the CNS” or “CNS disorder,” as those terms are usedherein, encompasses any condition that affects the brain and/or spinalcord and that leads to suboptimal function. In some embodiments, the CNSdisorder is an acute CNS disorder, such as brain injury, spinal cordinjury, focal brain ischemia and global brain ischemia. In embodimentsin which the disorder is an acute disorder, the composition isadministered only once. In embodiments in which the disorder is an acutedisorder, the composition is administered up to 10, 15, 20, 30, or morethan 30 times. In some embodiments the composition is administered at afrequency of no greater than about once per week. In some embodiments,the CNS disorder is a chronic disorder. In some embodiments, the chronicdisorder is selected from the group consisting of chronicneurodegenerative disease. In some embodiments where the disorder is achronic neurodegenerative disease, the chronic neurodegenerative diseaseis prion diseases, Alzheimer's disease, Parkinson's disease,Huntington's disease, multiple sclerosis. In some embodiments, thechronic disorder is selected from the group consisting of chronic braininfection. In some embodiments where the disorder is a chronic braininfection, the chronic infection is West Nile virus or humanimmunodeficiency virus. In some embodiments, the chronic disorder iscancer. In some embodiments where the disorder is a cancer, the canceris metastatic breast cancer to brain, metastatic cancer to brain, orprimary brain cancer.

In some embodiments, the invention provides methods of treatment of theretina, or for treatment or prevention of blindness. The retina, likethe brain, is protected from the blood by the blood-retinal barrier(BRB). The insulin receptor is expressed on both the BBB and the BRB,and the HIRMAb has been shown to deliver therapeutics to the retina viaRMT across the BRB. An antibody against the vascular endothelial growthfactor (VEGF) is protective in retinal disease, but it is necessary toinject the antibody directly into the eyeball, because antibody does notcross the BRB. In some embodiments, fusion proteins of the invention areused to treat retinal degeneration and blindness with a route ofadministration no more invasive than an intravenous or subcutaneousinjection, because the HIRMAb delivers the antibody pharmaceuticalacross the BRB, so that the antibody is exposed to retinal neural cellsfrom the blood compartment.

It will be appreciated that the compositions containing the antibodypharmaceuticals described herein may be further modified to transport atherapeutic substance to close proximity or contact with a pathologicalsubstance, e.g., such proximity or contact can be achieved by binding ofthe pharmaceutical antibody to the pathological substance. Such methodscan involve coupling of any suitable substance to the composition thatis capable of destroying or ameliorating the effect of the pathologicalsubstance while doing minimal or no damage to surrounding structures,e.g., an appropriate radionuclide, a toxin, or the like. Such methods ofcoupling and suitable substances are well-known in the art. It will befurther appreciated that damage to surrounding areas can be kept to aminimum if the substance is further transported out of the CNS acrossthe BBB as described herein.

Formulations and Administration.

Any suitable formulation, route of administration, and dose of thecompositions of the invention may be used. Formulations, doses, androutes of administration are determined by those of ordinary skill inthe art with no more than routine experimentation. Compositions of theinvention, e.g., fusion proteins are typically administered in a singledose, e.g., an intravenous dose, of about 0.01-1000 mg, or about0.05-500 mg, or about 0.1-100 mg, or about 1-100 mg, or about 0.5-50 mg,or about 5-50 mg, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15,20, 25, 30, 25, 40, 45, 50, 60, 70, 80, 90, or 100 mg. Typically, forthe treatment of acute brain disease, such as stroke, cardiac arrest,spinal cord injury, or brain trauma, higher doses may be used, whereasfor the treatment of chronic conditions such as Alzheimer's disease,Parkinson's disease, Huntington's disease, mad cow disease, MS, WestNile encephalitis, brain AIDS infection, or metastatic or primary braincancer, lower, chronic dosing may be used. Oral administration canrequire a higher dosage than intravenous or subcutaneous dosing,depending on the efficiency of absorption and possible metabolism of theprotein, as is known in the art, and may be adjusted from the foregoingbased on routine experimentation.

For intravenous or subcutaneous administration, formulations of theinvention may be provided in liquid form, and formulated in saline basedaqueous solution of varying pH (5-8), with or without detergents suchpolysorbate-80 at 0.01-1%, or carbohydrate additives, such mannitol,sorbitol, or trehalose. Commonly used buffers include histidine,acetate, phosphate, or citrate.

Dosages for humans can be calculated from appropriate animal data. Forexample, human dosing of a ScFv-MAb fusion protein is based onpre-clinical pharmacokinetic studies, and these measurements have beenperformed in Rhesus monkeys. The in vitro disaggregation assay, FIG. 41,shows that a concentration of 250 ng/mL of anti-Aβ ScFv/HIRMAb fusionprotein causes rapid disaggregation of amyloid plaque. The studies inRhesus monkey shows the brain volume of distribution of the anti-AβScFv/HIRMAb fusion protein is 100 uL/gram brain (FIG. 38). Therefore, inorder to achieve a brain concentration of 250 ng/gram brain, thecorresponding plasma concentration of the anti-Aβ ScFv/HIRMAb fusionprotein must be 5 ug/mL. The concentration in blood of the anti-AβScFv/HIRMAb fusion protein in a 7 kg primate is 0.05% ID/mL (FIG. 37).Therefore, the concentration in blood in a 70 kg human will be 0.005%I.D./mL. If the I.D.=100 mg in a human, then the blood level will be 5ug/ml and the concentration of the anti-Aβ ScFv/HIRMAb fusion protein inhuman brain will be 250 ng/g, which will cause rapid disaggregation ofamyloid plaque.

The antibody fusion protein can be formulated for chronic use for thetreatment of a chronic CNS disorder, e.g., neurodegenerative disease,stroke or brain/spinal cord injury rehabilitation, or depression.Chronic treatment may involve daily, weekly, bi-weekly administration ofthe composition of the invention, e.g., fusion protein eitherintravenously, intra-muscularly, or subcutaneous in formulations similarto that used for acute treatment. Alternatively, the composition, e.g.,fusion protein may be formulated as part of a bio-degradable polymer,and administered on a monthly schedule.

Combination Therapies.

The composition of the invention, e.g., fusion protein may beadministered as part of a combination therapy. The combination therapyinvolves the administration of a composition of the invention incombination with another therapy for the CNS disorder being treated. Ifthe composition of the invention is used in combination with another CNSdisorder method or composition, any combination of the composition ofthe invention and the additional method or composition may be used.Thus, for example, if use of a composition of the invention is incombination with another CNS disorder treatment agent, the two may beadministered simultaneously, consecutively, in overlapping durations, insimilar, the same, or different frequencies, etc. In some cases acomposition will be used that contains a composition of the invention incombination with one or more other CNS disorder treatment agents.

Other CNS disorder treatment agents that may be used in methods of theinvention include, without limitation, thrombolytic therapy for stroke,cholinergic-directed therapy for Alzheimer's disease, dopaminerestoration therapy for Parkinson's disease, RNA interference therapyfor genetic disorders, cancer, or infections, and anti-convulsanttherapy for epilepsy. Dosages, routes of administration, administrationregimes, and the like for these agents are well-known in the art.

In some embodiments, the composition, e.g., antibody fusion protein isco-administered to the patient with another medication, either withinthe same formulation or as a separate composition. For example, theantibody fusion protein could be formulated with another fusion proteinthat is also designed to deliver across the human blood-brain barrier arecombinant protein other than an anti-Aβ ScFv. The fusion protein maybe formulated in combination with other large or small molecules.

Compositions of the invention, e.g., fusion proteins, may be provided asa kit that includes the formulation, e.g., antibody fusion protein in acontainer and in suitable packaging. The composition can be provided ina dry powder form, in solid form (i.e., lyophilized), in solution, or insuspension. If the composition is a protein, to the proteins may havebeen added emulsifiers, salts, preservatives, other proteins, nucleicacids, protease inhibitors, antibiotics, perfumes, polysaccharides,adhesive agents, polymers, microfibrils, oils, etc. The composition ispackaged for transport, storage and/or use by a consumer. Such packagingof therapeutic compositions for transport, storage, and use iswell-known in the art. Packaged compositions may include furthercomponents for the dispensing and storage of the composition, and mayalso include separately packaged diluent comprised of, e.g., sterilewater or a suitable buffer, for solubilizing the formulation, e.g.,fusion protein prior to administration to the patient. Kits of theinvention may also include written materials, including instructions foruse, results of clinical studies, desired outcome and expected course oftreatment, information about precautions and side effects, and the like.The kits may optionally further contain other components, such asgloves, scissors, tape, implements for disposal of used vials and otherwaste, masks, antiseptic, antibiotics, and the like.

Abbreviations

9E10 MAb against 10-amino acid epitope of c-myc oncogene

AD Alzheimer's disease

ALS amyotrophic lateral sclerosis

anti-mAβScFv same as mAβScFv

AUC area under the plasma concentration curve

AUCss steady state AUC

Aβ amyloid peptide of AD

Aβ¹⁻⁴⁰ 40 amino acid Aβ amyloid peptide of AD

Aβ¹⁻⁴² 42 amino acid Aβ amyloid peptide of AD

Aβ¹⁻⁴³ 43 amino acid Aβ amyloid peptide of AD

BBB blood-brain barrier

BGH bovine growth hormone

Boc tert-butyloxycarbonyl

BRB blood-retinal barrier

CDR complementarity determining region

CED convection enhanced diffusion

CH1 first part of IgG constant region

CH2 second part of IgG constant region

CH3 third part of IgG constant region

CHO Chinese hamster ovary cell line

CLBA competitive ligand binding assay

CLss steady state systemic clearance

CMV cytomegalovirus

CNS central nervous system

COS CV-1 origin SV40 cell line

CT carboxyl terminus

Da Dalton

DHFR dihydrofolate reductase

DOTA 1,4,7,10-tetraazacyclododecane-N,—N′,N″,N′″-tetraacetic acid

DPM disintegrations per minute

E envelope protein of WNV

ECD extracellular domain

EDC N-methyl-N′-3-(dimethylaminopropyl)carbodiimide hydrochloride

EGF epidermal growth factor

EGFR EGF receptor

ELISA enzyme linked immunosorbant assay

FcR Fc receptor

FcRn neonatal FcR

FR framework region

FWD forward

HC heavy chain

HC-1 HC of chimeric HIRMAb

HD Huntington's disease

HGF hepatocyte growth factor

HIR human insulin receptor

HIRMAb monoclonal antibody to human insulin receptor

HIR-HC heavy chain (HC) of HIRMAb

HIR-LC light chain (LC) of HIRMAb

HIRMAb-mAβScFv fusion protein of HIRMAb and mAβScFv

HIV human immune deficiency virus

IC intra-cerebral

ICC immunocytochemistry

ICV intra-cerebroventricular

ID injected dose

IEF isoelectric focusing

IGF insulin-like growth factor

IgG immunoglobulin G

kb kilobase

IRMA immunoradiometric assay

LC light chain

LDL low density lipoprotein

MAb monoclonal antibody

mAβScFv murine ScFv against Aβ peptide

MRT mean residence time

MS multiple sclerosis

MTH molecular Trojan horse

MTX methotrexate,

MW molecular weight

NHS N-hydroxy succinimide

NK new Kozak sequence

NT amino terminus

NSP new signal peptide

nt nucleotide

ODN oligodeoxynucleotide

orf open reading frame

pA poly-A signal

PBS phosphate buffered saline

PCR polymerase chain reaction

PD Parkinson's disease

PET positron emission tomography

PNA peptide nucleic acid

PRO promoter

Prp prion protein

RE restriction endonuclease

REV reverse

RMT receptor mediated transport

RNAi RNA interference

RT reverse transcriptase

SA streptavidin

ScFv single chain Fv antibody

ScFv-MAb fusion antibody of a tetrameric MAb and a ScFv

SCLC small cell lung cancer

SDM site-directed mutagenesis

SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis

SFM serum free medium

SPECT single photon emission computed tomography

TAA tumor-associated antigen

TNF tumor necrosis factor

TRAIL TNF-related apoptosis-inducing ligand

TV tandem vector

VD volume of distribution

VEGF vascular endothelial growth factor

VH variable region of heavy chain

VL variable region of light chain

Vss steady state systemic volume of distribution

WNV West Nile virus

EXAMPLES

The following specific examples are to be construed as merelyillustrative, and not limitative of the remainder of the disclosure inany way whatsoever. Without further elaboration, it is believed that oneskilled in the art can, based on the description herein, utilize thepresent invention to its fullest extent. All publications cited hereinare hereby incorporated by reference in their entirety.

Example 1 Genetic Engineering of a Eukaryotic Expression PlasmidEncoding an Anti-Aβ ScFv

The genetic engineering of a eukaryotic expression vector encoding amouse single chain antibody to human Aβ peptide is outlined in FIG. 1.The final protein expression vector was designated pCD-mAβScFv (FIG.1B). This vector was designed to produce a single chain Fv antibody(ScFv), comprised of both the variable region of the heavy chain (VH)and the variable region of the light chain (VL) of a mouse (m)monoclonal antibody to human Aβ peptide; this ScFv is designatedmAβScFv. The VH and VL are fused by a 17 amino acid linker to form theScFv. The pCD-mAβScFv plasmid encodes the mAβScFv with a human IgGsignal peptide (FIG. 11; amino acid residues 1-19 of SEQ ID NO. 18), andits expression is driven by the CMV promoter. The pCD-mAβScFv expressionvector also encompasses a full Kozak sequence domain (i.e.GCCGCCACCATGG; nucleotides 732-744 of SEQ ID NO. 27) prior to the ATGmethionine initiation codon (ATG) (FIG. 1B). For the cloning of eitherthe VH and VL cDNA of the mAβScFv, poly A+ RNA was isolated from theoriginal murine hybridoma cell line and subjected to reversetranscription (RT) using oligo(dT)₁₂₋₁₈ and SuperScript II reversetranscriptase (Invitrogen, Carlsbad, Calif.) to form single strandedcomplementary DNA (scDNA). The VL cDNA was produced by polymerase chainreaction (PCR) using the VH or VL scDNA as template and the VL-specificforward (FWD) or reverse (REV) oligodeoxynucleotide (ODN) PCR primers(Table 2, SEQ ID NO. 1 and 2, respectively). VL forward and reverse ODNprimers introduce MluI and NotI restriction endonuclease (RE) sites,respectively, for directional cloning into the prokaryote ScFvexpression vector pAP-xScFv (FIG. 1A). The mAβVH cDNA was obtained byPCR using the mAβscDNA as template and the VH-specific forward andreverse ODN PCR primers (Table 2, SEQ ID NO. 3 and 4, respectively). VHforward and reverse ODN primers introduce NcoI and HindIII RE sites,respectively, for directional cloning into the prokaryote ScFvexpression vector pAP-xScFv (FIG. 1A). The PCR reactions were performedwith PfuUltra DNA polymerase (Stratagene, La Jolla, Calif.), and PCRproducts were resolved by agarose gel electrophoresis (FIG. 3A). Theexpected major cDNA bands corresponding to the PCR amplified ˜0.4kilobase (kb) mAβ VL cDNA and PCR amplified ˜0.4 kb mAβ VH cDNA areshown in FIG. 3A, lanes 1 and 2, respectively. The VH and VL bands wereisolated from the agarose gels and subcloned into the pPCR-Script toform pPC-mAβ-VH and pPC-mAβ-VL (FIG. 1A) using the PCR-Script AmpCloning Kit (Stratagene) for further characterization and DNAsequencing.

TABLE 2 Oligodeoxynucleotides used in the reverse transcription PCR cloning of the VH and VL domains of the mouse anti-Aβ antibody (mAβ), and  in the engineering of the HIR-mAβ fusion antibodyVL forward (SEQ ID NO. 1):5′-AATTTTCAGAAGCACGCGTAGATATC(G/T)TG(A/C)T(G/C) ACCCAA(A/T)CTCCA-3′VL reverse (SEQ ID NO. 2): 5′-GAAGATGGATCCAGCGGCCGCAGCATCAGC-3′VH forward (SEQ ID NO. 3):5′-CAGCCGGCCATGGCGCAGGT(G/C)CAGCTGCAG(G/C)AG-3′VH reverse (SEQ ID NO. 4): 5′-CCAGGGGCCAGTGGATAGACAAGCTTGGGTGTCGTTTT-3′Human IgG peptide signal FWD (SEQ ID NO. 5):5′ATCCTCGAGGCCGCCACCATGGACTGGACCTGGAGGGTGTTCTGCCTGCTTGCAGTGGCCCCCGGAGCCCACAGCCAGGTCCAGCTGCAG-3′Human IgG peptide signal REV (SEQ ID NO. 6):5′CTGCAGCTGGACCTGGCTGTGGGCTCCGGGGGCCACTGCAAGCAGGCAGAACACCCTCCAGGTCCAGTCCATGGTGGCGGCCTCGAGGAT-3′Human IgG peptide signal XhoI PCR FWD (SEQ ID NO.  7):5′-ATCCTCGAGGCCGCCACC-3′Human IgG peptide signal EcoRI PCR REV (SEQ ID NO.  8):5′-GATGAATTCTTATAGATCTTCTTCTGA-3′Mature mAβ ScFv PCR FWD (SEQ ID NO. 9):5′-phosphate-CACAGGTCCAGCTGCAGCAGT-3′Mature mAβ ScFv PCR REV (SEQ ID NO. 10):5′-phosphate-TTACCGTTTTATTTCCAGCTTGGTC-3′Human IgG peptide signal HIR FWD (SEQ ID NO. 31):CGAGCGGCCGCCACTGTGCTGGATATTCCACCATGGACTGGACCTGGAGGGTGTTCTGCCTGCTTGCAGTGGCCCCCGGAGCCCACAGCCAGGTTCAGCTGCAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTTAGTGAAGATAT CCTGHuman IgG peptide signal HIR REV (SEQ ID NO. 32):CAGGATATCTTCACTAAAGCCCCAGGCTTCACCAGCTCAGGTCCAGACTGCTGCAGCTGAACCTGGCTGTGGGCTCCGGGGGCCACTGCAAGCAGGCAGAACACCCTCCAGGTCCAGTCCATGGTGGAATATCCAGCACAGTGGCGGCCG CTCG

The nucleotide and deduced amino acid sequence of the mAβ VH are shownin FIGS. 4 (SEQ ID NO. 11) and 5 (SEQ ID NO. 12), respectively. The mAβVH cDNA sequence was 100% confirmed in several isolated clones, and itis compatible with the consensus sequence for the mouse gamma heavychain. The FR1-4 and CDR1-3 domain structure of the mAβ VH amino acidsequence in shown in FIG. 25. The nucleotide and deduced amino acidsequence of the mAβVL are shown in FIGS. 6 (SEQ ID NO. 13) and 7 (SEQ IDNO. 14), respectively. The mAβVH cDNA sequence was 100% confirmed inseveral isolated clones, and it is compatible with the consensussequence for the mouse kappa VL. The FR1-4 and CDR1-3 domain structureof the mAβVL amino acid sequence is shown in FIG. 25.

The amino acids comprising the CDR1, CDR2, and CDR3 of the anti-Aβ MAbVH are in bold font in FIG. 5, and correspond to amino acids 26-35,50-66, and 99-103, respectively of SEQ ID NO. 12. The amino acidscomprising the CDR1, CDR2, and CDR3 of the anti-Aβ MAb VL are in boldfont in FIG. 7, and correspond to amino acids 24-39, 55-61, and 94-102,respectively of SEQ ID NO. 14.

Both mAβ VH and VL cDNAs were subcloned into the prokaryote ScFvexpression vector pAP-xScFv to create the intermediate mAβ-ScFvexpression plasmid pAP-mAβScFv (FIG. 1A). The mAβ VH cDNA in pPC-mAβ-VHand the pAP-xScFv plasmid were digested with NcoI and HindIII. Theexpected mAβ-VH of ˜0.4 kb and the pAP-xScFv backbone of ˜3.5 kb (FIG.3B, lanes 1 and 2, respectively) were gel-purified. The mAβ-VH cDNA wasligated into the pAP-xScFv backbone at the same RE sites to form theintermediate plasmid pAP-mAβ-VH (FIG. 1A). The pAP-mAβ-VH plasmid wasisolated and characterized by DNA sequence, which confirmed 100% theexpected DNA sequence for mAβ-VH (FIG. 4, SEQ ID NO. 11). Theengineering of the pAP-mAβ-ScFv was completed by insertion of the mAβ-VLinto the pAP-mAβ-VH at MluI and NotI sites (FIG. 1A). Both pAP-mAβ-VHand the pPC-mAβ-VL clones were digested with MluI and NotI. The ˜0.4 kbmAβ-VL cDNA and the ˜3.5 kb pAP-mAβ-VH plasmid backbone (FIG. 3C, lanes1 and 2, respectively) were gel-purified. The mAβ-VL cDNA was ligatedinto pAP-mAβ-VH to form the pAP-mAβ-ScFv (FIG. 1A). The pAP-mAβ-ScFvplasmid was isolated and validated by DNA sequencing. The nucleotide anddeduced amino acid sequence of the pAP-mAβ-ScFv are shown in FIGS. 8(SEQ ID NO. 15) and 9 (SEQ ID NO. 16), respectively. The regionscorresponding to mAβ VH and VL cDNAs in pAP-mAβ-ScFv are 100% similar tothe ones of the individual VH and VL genes shown FIGS. 4 and 6,respectively. The pAP-xScFv prokaryote expression vector introduces ac-myc epitope (FIG. 9, amino acid residues 255-265) and a 5-histidine(His₅) tag (SEQ ID NO:69) at the end of the VL region (FIG. 9).

The engineering of the mAβ-ScFv eukaryotic expression vector, clonepCD-mAβ-ScFv, is summarized in FIG. 1B and it was performed by (a)deletion of the prokaryote PelB leader sequence in pAP-mAβ-ScFv, (b)insertion of a human IgG signal peptide including a full Kozak sequencedomain to form pAP-mAβ-ScFv-K, (c) PCR cloning of the mAβ-ScFv-K cDNA tointroduce XhoI and EcoRI RE sites, and (d) subcloning of thePCR-generated XhoI-mAβ-ScFv-K-EcoRI cDNA into the eukaryote expressionvector pCD to form the pCD-mAβ-ScFv plasmid. A DNA fragment of theprokaryote expression vector pAP-mAβ-ScFv, including the PelB leadersequence, was deleted with XhoI and PvuII, the latter located at thesecond amino acid of the mAβ-VH. The backbone vector was gel-purified.In parallel, ODNs corresponding to the artificial new human peptidesignal (Table 2, human IgG peptide signal FWD and REV ODNs,respectively, SEQ ID NO. 5 and 6) were annealed at 65° C. and purifiedwith Qiagen PCR extraction kit (Valencia, Calif.). The double stranded(ds) ODN was digested with XhoI and PvuII, purified with Qiagen PCRextraction kit and inserted at the same RE sites in thepAP-mAβ-ScFv-XhoI-PvuII vector to form the pAP-mAβ-ScFv-K intermediateplasmid (FIG. 1B). The dsODN inserts a full Kozak site and a human IgGsignal peptide (amino acid residues 1-19, SEQ ID NO. 18). Positiveclones were identified by RE mapping with EcoRI, a site that was presentin the deleted DNA fragment of the pAP-mAβ-ScFv, but absent in theinserted human IgG signal peptide sequence. The mAβ-ScFv cDNA wasfurther engineered to introduce XhoI and EcoRI RE sites by PCR fordirectional subcloning into the eukaryote expression vector pCD (FIG.1B). The PCR cloning of the mAβ-ScFv cDNA was performed with the humanIgG peptide signal XhoI PCR FWD and EcoRI PCR REV ODNs (Table 2, SEQ IDNO. 7 and 8, respectively). The REV ODN also introduces a TAA stop codonafter the c-myc tag of the vector (FIG. 10). Both the PCR products andthe pCD vector were digested with XhoI and EcoRI (FIG. 3D). The ˜5.4 kbpCD and the ˜0.8 kb mAβ-ScFv cDNA (FIG. 3D, lanes 1 and 2, respectively)were gel-purified and ligated at the same RE sites to form thepCD-mAβScFv expression vector (FIG. 1B). The pCD-mAβScFv clone wasvalidated by DNA sequencing, and both the nucleotide and deduced aminoacid sequences are shown in FIGS. 10 and 11 (SEQ ID NO. 17 and 18),respectively. The nucleotide and amino acid sequences corresponding tomAβ VH and VL cDNAs in pCD-mAβScFv (including the VH-VL linker) are 100%identical to the sequences in the pAP-mAβScFv vector shown FIGS. 8 and9, respectively. The pCD-mAβScFv vector does not have the His₅ tag (SEQID NO:69) (FIG. 11), so the TAA stop codon follows the end of the c-mycepitope (FIG. 10). The pCD-mAβScFv eukaryote expression plasmid isdriven by the CMV promoter, has a full Kozak domain before the ATGinitiation codon, and contains a human IgG signal peptide.

Example 2 Genetic Engineering of a Eukaryotic Expression PlasmidEncoding a Fusion Protein of the Anti-Aβ ScFv and the Chimeric HIRMAbHeavy Chain

The genetic engineering of the eukaryotic expression vector encoding forthe heavy chain of the fusion antibody was performed as summarized inFIG. 2. The fusion antibody heavy chain is comprised of the mAβScFv,which is fused at its amino terminus to the carboxyl terminus of theheavy chain (HC) of the chimeric HIRMAb; the HC of the chimeric HIRMAbis designated HC-1 in FIG. 2. The engineering of this gene encoding theHC of the fusion antibody was done in 2 steps: (a) PCR cloning of themAβScFv and (b) insertion of this cDNA into the universal HIR heavychain expression vector, designated pCD-UHC (FIG. 2), to form thepCD-HC-mAβScFv plasmid (FIG. 2). In the pCD-UHC expression vector, thechimeric HIRMAb heavy chain (HC) cDNA is preceded by the CMV promoterand is followed by the bovine growth hormone (BGH) polyadenylationsequence (pA). The pCD-UHC has a single HpaI site at the end of theHIRMAb HC open reading frame (orf) for insertion of genes of interestand expression of HIRMAb HC fusion proteins. For the PCR cloning of themature mAβScFv, the mature mAβ ScFv PCR FWD ODN (Table 2, SEQ ID NO. 9)was designed, so as to delete the human IgG peptide leader sequence fromthe mAβScFv cDNA, while maintaining the orf at the CH3 region of theHIRMAb heavy chain of the pCD-UHC. This results in the insertion of aSer-Ser linker between the end of the CH3 region of the HIR mAβ heavychain, and the mAβScFv cDNA. The mature mAβScFv REV PCR ODN (Table 2,SEQ ID NO. 10) was designed to delete the c-myc tag, and the linker[ADAAAAGS (amino acids 245-252, SEQ ID NO. 16; FIG. 9)], between the endof the mAβScFv cDNA and the c-myc tag, and to introduce a stop codon,TAA. Both PCR primers were 5′-phosphorylated for direct ligation intothe pCD-UHC at the HpaI site. The PCR cloning of the mature mAβScFv cDNAwas done using the pCD-mAβScFv DNA as template. Agarose gelelectrophoresis of the PCR products showed the expected single band of˜0.8 kb corresponding to the mature mAβScFv cDNA (lane 1, FIG. 3E). Theengineered mature mAβScFv was ligated at the HpaI site in pCD-UHC toform the pCD-HC-mAβScFv expression vector (FIG. 2). The pCD-HC-mAβScFvclone was validated by DNA sequencing, and both the nucleotide anddeduced amino acid sequences are shown in FIGS. 12 and 13 (SEQ ID NO. 19and 20), respectively. The nucleotide and amino acid sequence of thereconstructed carboxyl terminus at the CH3 region of the HIR mAb heavychain confirmed a 2-amino acid linker (Ser-Ser) prior to the maturemAβScFv and the TAA stop codon, both introduced in the PCR cloning step(FIGS. 12 and 13)

Example 3 I2V Site-Directed Mutagenesis of the FR1 of the VH of theAnti-Aβ ScFv

Amino acid microsequencing analysis of the amino terminus of the lightchain of the mAβ MAβ confirmed 11 residues, with the exception of theamino acid at position 2 of the light chain (FIG. 7). The PCR primersintroduced a isoleucine (I or Ile) at position 2, whereas the hybridomagenerated light chain contained a valine (V or Val) at position 2. TheIle residue was most likely introduced during the PCR cloning of the mAβVL due to the use of degenerate primers used in the PCR amplification ofthis cDNA. It was necessary to perform site-directed mutagenesis (SDM)to change the I residue to a V residue at position 2, and this SDM isdesignated the I2V change. A new eukaryotic expression vector encodingfor the heavy chain of the fusion antibody carrying the I2V VL mutantwas constructed. The SDM was performed using the QuickChange II XL SDMkit (Stratagene) and standard protocol. SDM-ODNs were designed tointroduced the mutation of interest, i.e. “A” for “G” at position 1789(FIG. 12, SEQ ID NO. 19). ODNs for SDM also contain 15 nucleotides ateach flanking region to anneal with the target sequence. SDM wascompleted using the pCD-HC-mAβScFv clone DNA as template to formpCD-HC-mAβScFv I2V, and the SDM clone was validated by DNA sequencing.The nucleotide and deduced amino acid sequences are shown in SEQ ID NOS:21 and 22, respectively. Both the nucleotide and deduced amino acidsequences of pCD-HC-mAβScFv-I2V are identical to the ones of theparental clone pCD-HC-mAβScFv (FIGS. 12 and 13) with the exception ofthe SDM-introduced mutation, which is A1789G (FIG. 14, underlined “G”residue) and I597V (FIG. 15, underlined “V” residue).

Example 4 Genetic Engineering of Signal Peptide and Full Kozak Site ofFusion Protein Heavy Chain

In order to optimize the expression of the fusion antibody heavy chain,the signal peptide (SP) of the chimeric HIRMAb heavy chain in pCD-UHC(FIG. 13, amino acid residues 1-19 of SEQ ID NO 20) was replaced by anew peptide signal, which had been used in the engineering ofpCD-mAβScFv (FIGS. 1B and 11, amino acid residues 1-19 of SEQ ID NO 18).The full Kozak consensus sequence was also introduced prior toengineering of the new pCD-HC-mAbScFv II plasmid (FIG. 2). The signalpeptide sequence of the chimeric HIRMAb expression vector, pCD-UHC, wasdeleted by double digestion with NotI and EcoRV, RE sites located in thepCD multiple cloning site and in the HIRMAb HC open reading frame. A DNAfragment comprised of 148 by was replaced by an artificial dsODN thatencompasses the human IgG peptide signal using the human IgG peptidesignal HIR FWD ODN (Table 2, SEQ ID NO. 31) and the human IgG peptidesignal HIR REV ODN (Table 2, SEQ ID NO. 32). Both the artificial dsODNand the pCD-UHC expression plasmid were digested with NotI and EcoRV andgel-purified. Engineering of the pCD-UHC with new signal peptide (NSP)continued with the ligation of the NSP into the pCD-UHC at the same REsites to form the pCD-UHC-NSP vector. The full Kozak consensus sequencewas then introduce by SDM using the QuickChange II XL SDM kit andstandard protocol. SDM-ODNs were designed to introduce the mutation ofinterest (i.e. GCCGCCACC) and also contain 15 nucleotides at eachflanking region to anneal with the target sequence. SDM was completedusing the chimeric HIRMAb heavy chain in pCD-HC-NSP DNA as template toform pCD-HC-NSP new Kozak (NK) vector. The pCD-HC-NSP-NK was convertedinto a Universal HIRMAb heavy chain expression vector for fusionproteins (pCD-UHC-II, FIG. 2) by insertion of a HpaI RE site at the stopcodon after the CH3 region by SDM. The SDM protocol was completed withthe QuickChange II XL SDM kit and SDM-ODNs to introduce the HpaI RE site(i.e. GTTAAC). ODNs also contain 15 nucleotides at each flanking regionto anneal with the target sequence. The genetic engineering of a neweukaryotic expression vector encoding for the heavy chain of the HIRMAbfusion antibody was performed using the optimized expression vectorpCD-UHC-II as summarized in FIG. 2. The engineering of new fusionantibody heavy chain fusion gene was done in 2 steps, (a) PCR cloning ofthe mAβScFv cDNA and (b) insertion of this cDNA into the new universalHIRMAb heavy chain expression vector pCD-UHC-II to form pCD-HC-mAβScFvII (FIG. 2). The mAβScFv cDNA was obtained by PCR cloning using thepCD-HC-mAβScFv-I2V (SEQ ID NO:21) as template and the mAb ScFv PCR FWDand REV ODNs (Table 2, SEQ ID NO. 9 and 10, respectively). Theengineered mAβScFv I2V cDNA was ligated at the HpaI site in pCD-UHC-IIto form the pCD-HC-mAβScFv II expression vector (FIG. 2). ThepCD-HC-mAβScFv II clone was validated by DNA sequencing, and both thenucleotide and deduced amino acid sequences are shown in FIGS. 14 and 15(SEQ ID NO. 21 and 22), respectively. The nucleotide and amino acidsequence of the reconstructed carboxyl terminus at the CH3 region of theHIRMAb heavy chain confirmed a 2-amino acid linker (Ser-Ser) prior tothe mAβScFv I2V and the TAA stop codon, both introduced in the PCRcloning step (FIGS. 14 and 15). The NSP was also confirmed, i.e.nucleotides 1-57 (FIG. 14; SEQ ID NO 21) and amino acids 1-19 (FIG. 15;SEQ ID NO 22).

Example 5 N497A Site-Directed Mutagenesis of the CDR2 of the VH of theAnti-Aβ ScFv

The mAβScFv has a predicted variable region N-glycosylation domain inthe second CDR of the VH, which is underlined in FIG. 25, and whichcorresponds to the asparagine (N or Asn) residue at position 497 of theVH without the signal peptide shown in FIG. 24; if the signal peptide ofthe mAβScFv is included, this Asn residue corresponds to position 516 ofFIG. 15 and SEQ ID NO. 22. Because glycosylation of the variable regionof an antibody may affect binding, the N-glycosylation domain of themAβScFv was mutated in clone pCD-HC-mAbScFv II, which corresponds to SEQID NO. 21, to form new clone named pCD-HC-mAbScFv II-N497A; the mAβScFvpart of the heavy chain of the fusion antibody produced by this cloneexpresses an alanine (A or Ala) residue at position 497 instead of theasparagine, and this SDM is designated N497A. The SDM of residue N497Awas completed with standard protocol and the QuickChange II XL SDM kit.SDM-ODNs were designed to introduce the mutation of interest, i.e. “GC”nucleotides at positions 1546-1547 (FIG. 16, SEQ ID NO. 23) and contain15 nucleotides at each flanking region to anneal with the targetsequence. SDM was competed using the pCD-HC-mAbScFv II clone DNA astemplate (SEQ ID NO. 21), and the SDM clone was validated by DNAsequencing. The nucleotide and deduced amino acid sequences are shown inFIGS. 16 and 17 (SEQ ID NO. 23 and 24), respectively. Both thenucleotide and deduced amino acid sequences of pCD-HC-mAβScFv II N497Aare identical to the ones of the parental clone pCD-HC-mAβScFv II withthe exception of the SDM-introduced mutations, i.e. 1546GC1547 (FIG. 16,underlined “GC” residues) and N497A (FIG. 17, underlined “A” residue).COS cells were dual transfected with pCD-HC-mAbScFv II-N497A plasmid,and the pCD-LC plasmid, where pCD-LC is a eukaryotic expression plasmidencoding the light chain of the HIRMAb. The fusion antibody with theN497A mutation was purified from the COS cell conditioned medium byprotein A affinity chromatography. The affinity of the Ala-497 form ofthe antibody fusion protein for the Aβ¹⁻⁴⁰ peptide was measured with animmunoradiometric assay (IMRA) as described below in Example 10. TheIRMA showed the affinity of the Ala-497 fusion antibody for the Aβ¹⁻⁴⁰was characterized by a K_(I) of 182±32 nM, which is decreased nearly6-fold, as compared to the Asn-497 fusion antibody shown in FIG. 34 andExample 10. Therefore, the Asn residue at position 497 was left intactin all future investigations.

Example 6 S499A Site-Directed Mutagenesis of the CDR2 of the VH of theAnti-Aβ ScFv

As an alternative strategy to mutation of the variable regionglycosylation site in the mAβScFv the serine residue (S or Ser) atposition 499 was mutated to an alanine residue, and this mutation isdesignated the S499A mutation. The latter was mutated in clonepCD-HC-mAbScFv II (SEQ ID NO. 21) to form new clone named pCD-HC-mAbScFvII S499A. The SDM of this residue was completed with standard protocoland the QuickChange II XL SDM kit. SDM-ODNs were designed to introducethe mutation of interest, i.e. “GC” at positions 1552-1553 (FIG. 14, SEQID NO. 21) and contain 15 nucleotides at each flanking region to annealwith the target sequence. SDM was competed using the pCD-HC-mAβScFv IIclone DNA as template, and the SDM clone was validated by DNAsequencing. The nucleotide and deduced amino acid sequences are shown inFIGS. 18 and 19 (SEQ ID NO. 25 and 26), respectively. Both thenucleotide and deduced amino acid sequences of pCD-HC-mAβScFv II S499Aare identical to the ones of the parental clone pCD-HC-mAβScFv II withthe exception of the SDM-introduced mutations, i.e. 1552GC1553 (FIG. 18,underlined “GC” residues) and S499A (FIG. 19, underlined “A” residue).COS cells were dual transfected with pCD-HC-mAbScFv II-S499A plasmid,and the pCD-LC plasmid. The secretion of the fusion antibody by thetransfected cells was assayed by measurement of human IgG secreted tothe medium. The level of secretion of the fusion antibody was unchangedfollowing engineering of the S499A mutation. The fusion antibody withthe S499A mutation was purified from the COS cell conditioned medium byprotein A affinity chromatography. The affinity of the Ala-499 form ofthe antibody fusion protein for the Aβ¹⁻⁴⁰ peptide was measured with animmunoradiometric assay (IMRA) as described below in Example 10. TheIRMA showed the affinity of the Ala-499 fusion antibody for the Aβ¹⁻⁴⁰was characterized by a K_(I) of 271±119 nM, which is decreased 8-fold,as compared to the Ser-499 fusion antibody shown in FIG. 34 and Example10. Therefore, the Ser residue at position 499 was left intact in allfuture investigations.

Example 7 Substitution of Constant Region, Including Site-DirectedMutagenesis of Constant Region Glycosylation Site

Similar to Examples 5 and 6, it is possible to perform site-directedmutagenesis of the consensus glycosylation site in the constant (C)region of the heavy chain of the fusion antibody. This consensussequence is NST (Asn-Ser-Thr) of the CH2 region, which is underlined inFIGS. 21 and 25. Substitution of either the Asn or the Ser or the Thrresidue of this sequence can abolish the C-region glycosylation. Removalof the glycosylation of the C-region should have no effect on binding ofthe fusion antibody to either the HIR or the target antigen such as theAβ peptide. Removal of the C-region carbohydrate has no effect onbinding of IgG to Fc receptors (FcR), such as the neonatal FcR, alsocalled the FcRn. The constant region used in the present examples is theC-region from the human IgG1 subclass. This C-region includes thehinge-CH1-CH2-CH3 regions shown in FIG. 25, in encompasses amino acidresidues 133-462 of SEQ ID NO. 28. The IgG1 C-region could besubstituted with the C-region of human IgG2, IgG3, or IgG4. Thesub-domains, hinge, CH1, CH2, CH3, or CH4, could be interchanged betweenthe different IgG subclasses.

Example 8 Genetic Engineering of Tandem Vector Expressing AntibodyFusion Protein

The genetic engineering of the eukaryotic expression tandem vectorencoding for the fusion antibody was performed as summarized in FIG. 24.The tandem vector is comprised of several expression cassettesincluding: (a) the HIRMAb light chain (LC), (b) the HIRMAb heavy chain(HC) and (c) the dihydrofolate reductase (DHFR) expression cassettes.Both the LC and HC genes are driven by the CMV promoter and contain BGHpolyadenylation sequences and human IgG signal peptide. The DHFR gene isregulated by the SV40 promoter and contains the hepatitis Bpolyadenylation sequence. All 3 genes have full Kozak sequences. Theengineering of this fusion gene was done in 2 steps: (a) PCR cloning ofthe mAβScFv, and (b) insertion of this cDNA into the tandem vector tocreate the fusion antibody tandem vector (FIG. 24). The mAβScFv cDNA wasobtained by PCR cloning using the pCD-HC-mAbScFv II (SEQ ID NO.21) astemplate and the mAβScFv PCR FWD and REV ODNs (Table 2, SEQ ID NO. 9 and10, respectively). The engineered mature mAbScFv cDNA was ligated at theHpaI site of the tandem vector to form the fusion antibody tandem vector(FIG. 24). The fusion antibody tandem vector was validated by DNAsequencing, and the nucleotide sequence is shown in FIG. 20 (SEQ ID NO.27). The deduced amino acid sequences for the fusion antibody HC, theHIRMAb LC, and DHFR genes are shown in FIGS. 21-23 (SEQ ID NO. 28-30),respectively. The nucleotide and amino acid sequence of thereconstructed carboxyl terminus at the CH3 region of the HIRMAb heavychain confirmed a 2-amino acid (Ser-Ser) linker, preceding the mAβScFvand the TAA stop codon, following the mAβScFv, and both modificationswere introduced in the PCR cloning step (FIG. 24). The LC expressioncassette is contained within nucleotides (nt) 1-1736 of FIG. 20 (SEQ IDNO 27), and is comprised of (a) a CMV promoter, nt 1-731, (b) a fullKozak sequence, nt 732-740, (c) the LC orf, nt 741-1445, and (d) a BGHpolyadenylation sequence, nt 1446-1736. The HC expression cassette iscontained within nt 1760-4904 of FIG. 20 (SEQ ID NO 27), and iscomprised of (a) a CMV promoter, nt 1760-2473, (b) a full Kozaksequence, nt 2474-2482, (c) the HC orf, nt 2483-4609, and (d) a BGHpolyadenylation sequence, nt 4610-4904. The DHFR expression cassette iscontained within nt 4905-6671 of FIG. 20 (SEQ ID NO 27), and iscomprised of (a) a SV40 promoter, nt 4905-5158, (b) a full Kozaksequence, nt 5159-5167, (c) the DHFR orf, nt 5168-5731, and (d) ahepatitis B polyadenylation sequence, nt 5732-6671.

The fusion antibody tandem vector (FIG. 24) was linearized with PvuI andelectroporated into CHO DG44 cells followed by selection with G418 (500ug/ml) and hypoxanthine-thymidine deficient medium for 3 weeks. Positiveclones were detected in 96 well plates with a human IgG ELISA that uses2 primary antibodies to both the human IgG1 HC and the human kappa LC.Cell lines of high copy number of the transgene were selected by gradedincreases in MTX to 600 nM. The G418/MTX selected cell lines weremaintained in high density and continued to secrete human IgG. Thetransfected CHO cells were subjected to a round of limited dilutionalcloning, and produced human IgG at a level of 10 mg/L in shake flasks.Following affinity purification the CHO cell derived fusion protein wasanalyzed by SDS-PAGE and human IgG Western blotting, and the fusionheavy chain and light chain migrated identical to that observed for thefusion antibody produced in COS cells and shown in FIG. 32.

Example 9 Diverse Structural Domains of Antibody Fusion Protein

The heavy chain of the fusion antibody is comprised of 28 domains asshown in FIG. 25. The 19-amino acid human IgG signal peptide is cleavedin the secretion of the fusion antibody from the intracellularcompartment. The constant region of human IgG is comprised of 4 domains:CH1, hinge, CH2, and CH3. The variable region of the heavy chain (VH) ofthe chimeric HIRMAb is fused to the amino terminus of CH1; this VH iscomprised of 4 framework regions (FR), designated FR1, FR2, FR3, andFR4, and 3 complementarity determining regions (CDR), designated CDR1,CDR2, and CDR3. A serine-serine (S-S) linker joins the carboxyl terminusof CH3 with the amino terminus of the VH of the anti-Aβ ScFv; the VH andthe VL of the ScFv are also comprised each of 4 FR and 3 CDR regions.The VH and the VL of the anti-Aβ ScFv are joined by a 17-amino acidlinker. This linker is formed by amino acid sequences from the humanα-tubulin protein (Genbank CAA25855) to reduce immunogenicity in humans.The heavy chain shown in FIG. 25 covalently binds to the light chain ofthe chimeric HIRMAb, and to another fusion antibody heavy chain viadisulfide bridges in the hinge region to form the hetero-tetramericstructure shown in FIG. 26. The fusion antibody depicted in FIG. 26possesses 3 functionalities, each of which exerts a specific action inthe clearance of Aβ amyloid aggregates in the brain of AD. As shown inFIG. 27, there are 3 steps in the clearance of aggregated protein fromthe blood. First, influx of the fusion antibody across the BBB fromblood to brain via the HIR expressed at the BBB; this step is mediatedby the HIRMAb part of the fusion antibody, or the “head” of the moleculeshown in FIG. 26. Second, binding and disaggregation of the aggregatedprotein in brain; this step is mediated by the anti-Aβ ScFv part of thefusion antibody, or the “tail” of the molecule shown in FIG. 26. Third,efflux of the fusion antibody/aggregate protein complex from brain toblood across the BBB via the FcR expressed at the BBB; this step ismediated by binding of the CH2-CH3 parts of the constant region of thefusion antibody, or the “mid-section” of the molecule shown in FIG. 26.

Example 10 Eukaryotic Expression and Characterization of Anti-Aβ ScFv

COS-1 cells were grown in serum free medium and transfected withpCD-mAβScFv (FIG. 1B) using Lipofectamine-2000. The conditioned mediumwas removed at 3 or 7 days. The medium conditioned by COS cellstransfected with pCD-mAβScFv and Lipofectamine-2000 was concentratedwith an Ultra-15 (Amicon) filtration unit with a 10 kDa molecular weightcutoff, and was solubilized in sodium dodecyl sulfate (SDS) samplebuffer under reducing conditions, and applied to a 15%SDS-polyacrylamide gel for SDS-polyacrylamide gel electrophoresis (PAGE)followed by Western blotting with the 9E10 MAb. The 9E10 MAb binds to anepitope derived from the c-myc protein, and this epitope, EQKLISEEDL(SEQ ID NO: 66), is present at the carboxyl terminus of the anti-Aβ ScFv(FIG. 11). The positive control in the Western blot (FIG. 28, lane 1) isthe OX26 ScFv/streptavidin (SA) fusion protein, which was affinitypurified from bacterial pellets. The OX26 ScFv/SA fusion protein iscomprised of 3 domains: (i) the 29 kDa OX26 ScFv, (ii) the 16 kDa SAmonomer, and (iii) the C-terminal 10-amino acid c-myc epitope, whichreacts with the 9E10 MAb. The negative control in the Western blot (FIG.28, lane 2) is media from COS cells exposed to Lipofectamine 2000, butno plasmid DNA. The anti-Aβ ScFv lacks the SA domain and is comprised of(i) the anti-Aβ ScFv (27 kDa), and (ii) the C-terminal 10-amino acidc-myc epitope (2 kDa), which reacts with the 9E10 MAb. The 9E10 MAb alsocross-reacts with 2 proteins of 35-37 kDa that are secreted bynon-transfected COS cells (FIG. 28, lane 2). The 29 kDa anti-Aβ ScFv isspecifically secreted to the medium by the COS cells transfected withpCD-mAβScFv (FIG. 28, lane 3). This Western blot studied verified thatthe mAβScFv was secreted intact by the COS cells transfected with thepCD-mAβScFv.

The binding of the mAβScFv to the Aβ¹⁻⁴⁰ amyloid peptide was verifiedwith a specific ELISA. The Aβ¹⁻⁴⁰ amyloid peptide was plated in 96-wellplates, followed by the addition of the media conditioned by COS cellstransfected with the pCD-mAβScFv plasmid and Lipofectamine-2000. Theanti-Aβ ScFv contains a C-terminal 10-amino acid c-myc epitope, which isrecognized by the 9E10 MAb (FIG. 29A). The 9E10 MAb is biotinylated,which enables quantitation of the sc165 binding to Aβ¹⁻⁴⁰ by aperoxidase detection system and A492 readings (FIG. 29A). This ligandbinding assay showed the anti-Aβ ScFv binds well to the Aβ¹⁻⁴⁰ amyloidpeptide, whereas there is no signal when COS cell media is obtained fromcells exposed only to Lipofectamine-2000 (FIG. 29B).

The binding of the anti-Aβ ScFv to the amyloid plaque of AD was verifiedwith immunocytochemistry and sections of autopsy AD human brain. Theconcentrated conditioned medium obtained from COS cells transfected withthe pCD-mAβScFv was used to test the functional activity of the anti-AβScFv, with respect to binding to the Aβ plaque of AD. Frozen AD brainwas used to prepare 10 um frozen sections, which were fixed in 2%paraformaldehyde. The COS cell medium was co-incubated with the 9E10MAb, which is a murine MAb that binds the 9E10 epitope of the anti-AβScFv, and the mixture was applied to the AD frozen sections. This 9E10MAb will bind the c-myc epitope at the C-terminus of the anti-Aβ ScFv,similar to the Western blotting and binding assay format (FIG. 29A). Thesecondary antibody was a biotinylated horse-anti-mouse IgG, which bindsthe 9E10 MAb, a mouse IgG1. The anti-Aβ ScFv strongly stained theamyloid plaque of autopsy AD sections, as shown in FIGS. 30A and 30C. Noimmune staining of amyloid plaque was observed with the negativecontrols, which included the 9E10 MAb plus medium conditioned by COScells exposed to lipofectamine 2000 but without transfection withpCD-mAβScFv (FIG. 30B), or mouse IgG1, which is the isotype control ofthe 9E10 MAb (FIG. 30D). The Aβ ligand binding assay and the ADimmunocytochemistry (FIGS. 29-30) both show the anti-Aβ ScFv avidlybinds the Aβ amyloid of Alzheimer's disease, and that this anti-Aβ ScFvcould be used to produce a fusion protein with the chimeric HIRMAb, asoutlined in FIG. 26.

Example 11 Eukaryotic Expression and Characterization of Anti-AβScFv/Chimeric HIRMAb Fusion Protein

COS cells were dual transfected with the pCD-HC-mAβScFv (FIG. 2), whichis the fusion protein heavy chain expression plasmid and with pCD-LC,which is the HIRMAb light chain expression plasmid using Lipofectamine2000. Following 4 days of culture, the medium was harvested, and theAβScFv/chimeric HIRMAb fusion protein was purified by protein A affinitychromatography. The processing of the fusion antibody was examined byWestern blotting, and the bifunctionality of the fusion antibody wasexamined with ligand binding assays directed at either the HIR orAβ¹⁻⁴⁰. The fusion antibody and the chimeric HIRMAb was subjected toSDS-PAGE under reducing conditions, and the gel was stained withCoomasie blue (FIG. 31). These results show the fusion antibody waspurified to homogeneity on SDS-PAGE, and that the size of the lightchain (LC) for both the fusion antibody and the chimeric HIRMAb areidentical in size, as expected (FIG. 31). The heavy chain (HC) of thefusion antibody is 82 kDa, whereas the size of the HC of the chimericHIRMAb is 55 kDa (FIG. 31). The difference in size, 27 kDa, is due tothe fusion of the AβScFv to the HC of the fusion antibody.

The SDS-PAGE was repeated, and following blotting to nitrocellulose, theblot was probed with a primary antibody to human IgG. The antibodydetected identical size 28 kDa light chains in both the chimeric HIRMAband the fusion antibody (FIG. 32), which is expected because the ScFv isfused to the heavy chain (FIG. 25). The size of the chimeric HIRMAbheavy chain was the expected 55 kDa (FIG. 32). The size of the fusionantibody heavy chain was 82 kDa (FIG. 32), which is the sum of the 55kDa chimeric HIRMAb heavy chain, and the 27 kDa anti-Aβ ScFv.

The isoelectric point (pI) of the fusion antibody, the chimeric HIRMAb,and the hybridoma generated anti-Aβ MAb was determined by isoelectricfocusing (IEF), as shown in FIG. 33. The pI of the chimeric HIRMAb andthe fusion protein wee nearly identical, about 8.5, whereas the pI ofthe murine anti-Aβ MAb was more acidic with a pI of about 6.8. Thetheoretical pI of the fusion antibody heavy chain is predicted to be8.8, which matches the experimentally observed pI in FIG. 33.

The affinity of the fusion antibody for binding to Aβ¹⁻⁴⁰ was comparedto the same affinity of the murine hybridoma generated anti-Aβ MAb withan immunoradiometric assay (IMRA). In this assay, the Aβ¹⁻⁴⁰ is platedin 96-well plates, and the binding of [¹²⁵I]-murine anti-Aβ MAb to theAβ¹⁻⁴⁰ is measured. The dissociation constant, K_(D), of the murineanti-Aβ MAb binding to the Aβ¹⁻⁴⁰ is 32±11 nM (FIG. 34). The K_(D) offusion protein binding to the Aβ¹⁻⁴⁰ is 24±4 nM (FIG. 34). Therefore,the affinity of the fusion antibody for Aβ¹⁻⁴⁰ is identical to that ofthe original 150 kDa hetero-tetrameric murine anti-Aβ MAb. This was asurprising finding, since the affinity of a ScFv for the target antigenis generally much lower than for the full, tetrameric MAb molecule.Owing to the bivalency of the tetrameric MAb, the affinity for theantigen is higher than the affinity of the monomeric ScFv. The highaffinity of the ScFv moiety of the fusion antibody for Aβ¹⁻⁴⁰ isattributed to the design of the fusion antibody molecule, which placesthe ScFv in a dimeric or bivalent conformation (FIG. 26).

The affinity of the fusion antibody for binding to the human insulinreceptor (HIR) extracellular domain (ECD) was measured with an ELISAusing affinity purified HIR ECD obtained from medium conditioned by CHOcells permanently transfected with the HIR ECD gene. As shown in FIG.35, both the chimeric HIRMAb and the fusion antibody bind the HIR ECDwith high affinity. The 50% saturation of binding, ED₅₀, is 0.53±0.02 nMfor the chimeric HIRMAb. The ED₅₀ of fusion antibody binding to the HIRis 1.0±0.1 nM (FIG. 35). Therefore, the affinity of fusion antibody forthe HIR is >50% of the affinity of the original chimeric HIRMAb. The HIRat the BBB is an heterotetrameric molecule comprised of two alpha andtwo beta chains. The binding of the [¹²⁵I]-fusion antibody to the intactHIR at the human BBB was demonstrated with a radio-receptor assay usingisolated human brain capillaries (FIG. 36A), as an in vitro model of thehuman BBB. There is specific binding of the [¹²⁵I]-fusion antibody tohuman brain capillaries in a time-dependent process, whereas binding ofthe [¹²⁵I]-mouse anti-Aβ antibody is constant with time, and isnon-specific (FIG. 36B).

Example 12 Influx Across the BBB from Blood to Brain of Anti-AβScFv/Chimeric HIRMAb Fusion Antibody in Adult Rhesus Monkey In Vivo

The fusion antibody was iodinated with [¹²⁵I]-iodine and chloramine T toa specific activity of 19 μCi/μg. In parallel, the murine anti-Aβ MAbwas tritiated with [³H]-N-succinimidyl proprionate to a specificactivity of 0.42 uCi/ug. A 8 year old female Rhesus monkey, weighing10.2 kg, was administered by a single intravenous injection a dose of777 μCi of [¹²⁵I]-fusion antibody and 888 uCi of [³H]-murine anti-AβMAb. Serum was collected at multiple time points over a 180 min period.The serum glucose of the anesthetized, overnight-fasted primate wasconstant throughout the 180 min study period, and averaged 90±2 mg %,which indicates that the administration of the fusion antibody caused nointerference of the endogenous insulin receptor, and had no effect onglycemia control.

The serum removed from the anesthetized Rhesus monkey was analyzed fortotal radioactivity, and expressed as a % of injected dose (I.D.)/mLserum (FIG. 37). The ¹²⁵I radioactivity was counted in a gamma counter,and the ³H radioactivity was counted in a liquid scintillation counter;the ¹²⁵I isotope emits radioactivity in the ³H window and standardcurves were prepared to eliminate ¹²⁵I spill-over into the ³H channel.The serum % I.D./mL for the [³H]-murine anti-Aβ MAb was constant at alltime points, and averaged 0.25% I.D./mL (FIG. 37). The constant bloodconcentration of the [³H]-murine anti-Aβ MAb indicated this MAb was notsignificantly cleared by the primate tissues in vivo, which isconsistent with the known prolonged blood mean residence time (MRT) ofmonoclonal antibodies. In contrast, the serum % I.D./mL for the[¹²⁵I]-fusion antibody decreased rapidly to about 0.05% I.D./mL (FIG.37), which is indicative of rapid clearance of the fusion antibody fromblood via tissues expressing the insulin receptor. Although the HIRMAbdoes not react with the rodent insulin receptor, the HIRMAb does crossreact with the insulin receptor in Old World primates such as the Rhesusmonkey.

The serum concentration profile for the [¹²⁵I]-fusion antibody was fitto a 2-compartment pharmacokinetic (PK) model to yield thepharmacokinetics parameters listed in Table 3. The PK parameters for the[¹²⁵I]-fusion antibody are compared in Table 3 to the PK parameters inthe adult Rhesus monkey for [¹¹¹In]-chimeric HIRMAb. The systemicclearance rate of the fusion antibody is no different from that of thechimeric HIRMAb, which indicates fusion of the ScFv to the heavy chainof the HIRMAb does not alter systemic clearance. Systemic clearance ofeither the chimeric HIRMAb or the fusion antibody is a function ofantibody uptake by peripheral tissues, e.g. liver or spleen, whichexpress high amounts of insulin receptor at the vascular barrier of thetissue. In fact, the CNS is virtually the only organ where themicrovascular endothelium expresses significant amounts of insulinreceptor. Clearance of the HIRMAb or the fusion antibody by organs, e.g.heart or skeletal muscle, that are perfused by capillaries withcontinuous endothelium that does not express insulin receptor, would notbe expected to clear significant amounts of an antibody directed againstthe insulin receptor. In the case of liver or spleen, these organs areperfused by sinusoidal capillary compartments that are freely permeableto large molecules such as monoclonal antibodies. The HIRMAb or fusionantibody is rapidly exposed to the insulin receptor on parenchymal cellsin liver or spleen, and uptake into these organs accounts for the rapiddecrease in serum concentration of the HIRMAb or fusion antibody afterintravenous injection (FIG. 37).

TABLE 3 Pharmacokinetic parameters for [¹²⁵H]-fusion antibody and[¹¹¹In]-chimeric HIRMAb [¹¹¹In]-chimeric Parameter HIRMAb [¹²⁵I]-fusionantibody A₁ (% ID/ml) 0.15 ± 0.01 0.11 ± 0.01 A₂ (% ID/ml) 0.10 ± 0.010.048 ± 0.019 k₁ (min⁻¹) 0.12 ± 0.02 0.16 ± 0.02 k₂ (min⁻¹) 0.0018 ±0.0010 0.00090 ± 0.00033 t_(1/2) ¹ (min) 5.8 ± 0.6 4.4 ± 0.4 t_(1/2) ²(min) 380 ± 39  769 ± 282 Vss (ml/kg) 116 ± 11  200 ± 9  AUCss (%IDmin/ml) 55 ± 5  54 ± 16 CL_(SS) (ml/min/kg) 0.22 ± 0.08 0.18 ± 0.05MRT (hours) 8.9 ± 0.9 18.2 ± 6.7  A₁, A₂, k₁, and k₂ are the interceptsand slopes of the bi-exponential function describing the decay in plasmaconcentration with time. t_(1/2) ¹ and t_(1/2) ² are computed from k₁and k₂, respectively, and are the half-times of the decay curves foreach exponent. CL_(ss) AUCss, Vss, and MRT are the steady stateclearance, steady state area under the serum concentration curve, steadystate systemic volume of distribution, and mean residence time,respectively, and are computed from A₁, A₂, k₁, and k₂ using standardpharmacokinetic formulations.

At 180 minutes after drug injection, the animal was euthanized, andbrain radioactivity was analyzed with the capillary depletion method(FIG. 38). This method separates brain homogenate into a capillarypellet and a post-vascular supernatant. If the volume of distribution(VD) of the antibody in the post-vascular supernatant is high, then thisis evidence that the antibody has crossed the BBB and entered into thebrain interstitial and intracellular spaces. The VD has units of uL/grambrain and is the ratio of the concentration of the antibody in brain(DPM/g) divided by the concentration of the antibody in serum (DPM/uL)at the 180 terminal time point. The brain VD of the [³H]-murine anti-AβMAb is 10 uL/gram brain in either the homogenate or the post-vascularsupernatant (FIG. 38), and this VD is equal to the brain plasma volume.Therefore, the low VD of the [³H]-murine anti-Aβ MAb is evidence thatthis MAb, similar to MAb's in general, does not cross the BBB. That is,the murine anti-Aβ MAb, in either the murine form, a chimeric form, or ahumanized form, would not cross the human BBB, and could not be used asan amyloid clearing therapeutic for AD. The failure of the anti-Aβ MAbto cross the BBB, as shown by the data in FIG. 38, means this antibodytherapeutic could not be developed as a drug for the diagnosis ortreatment of AD. Similarly, other anti-Aβ MAb molecules do not cross theBBB, which is why there is no MAb-based therapeutic approved for thetreatment of AD. In contrast, the fusion antibody rapidly crosses theprimate BBB, as demonstrated by the high VD shown in FIG. 38. Furtherevidence that the fusion antibody freely crosses the BBB, and enters allparts of brain is the 3 hour brain scan of radioactivity in the Rhesusmonkey brain (FIG. 39). The high brain uptake of the fusion antibody isdue to the ability of this molecule to bind to the BBB insulin receptorfrom the blood compartment, and this binding to the BBB insulin receptortriggers receptor-mediated transport into the brain. The brain uptake ofthe fusion antibody is higher in gray matter, as compared to whitematter, as shown in FIG. 39, because the vascular density in gray matteris much higher than in white matter of brain.

The in vivo brain uptake in Rhesus monkey shown in FIGS. 38-39, and theHIR binding assay in FIG. 35, indicates the fusion antibody is able toinflux across the BBB in the blood to brain direction via the BBBinsulin receptor. Therefore, the fusion antibody is shown to performstep 1 in the scheme outlined in FIG. 27. The fusion antibody is alsoable to perform steps 2 and 3 of the scheme in FIG. 27, as illustratedby the following examples.

Example 13 Efflux Across the BBB from Brain to Blood of Anti-AβScFv/Chimeric HIRMAb Fusion Protein Mediated Via Fc Receptor in AdultRat Brain In Vivo

The [¹²⁵I]-fusion antibody (0.03 uCi in 0.3 uL) was injected into thecortex of the brain of the anesthetized adult rat under stereotaxicguidance per the standard protocol of the Brain Efflux Index technique.See, e.g., Zhang, Y. and Pardridge, W. M. (2001): Mediated efflux of IgGmolecules from brain to blood across the blood-brain barrier. JNeuroimmunol, 114: 168-172, incorporated herein by reference. The fusionantibody was injected in the par2 region of the parietal cortex ofbrain, with the following stereotaxic coordinates: 0.2 mm anterior tobregma; 5.5 mm lateral to bregma; 4.5 mm deep from the dural surface.This region is far removed from the cerebrospinal fluid (CSF) tracts,and efflux of radioactivity from brain over time can only occur viaefflux across the BBB from brain to blood. The rate of efflux of the[¹²⁵I]-fusion antibody from rat brain was followed over the next 90minutes. During this time >50% of the injected dose of the [¹²⁵I]-fusionantibody had effluxed from brain (FIG. 40). In contrast, other largemolecules efflux from rat brain with a half-time of about 10 hours. Therapid efflux of IgG molecules from brain is mediated by the BBB Fcreceptor (FcR), including the neonatal form of FcR, also called theFcRn. The BBB FcR mediates the asymmetric efflux of IgG from brain toblood, but not the influx of IgG from blood to brain. The efflux of the[¹²⁵I]-fusion antibody from brain is mediated by the BBB FcR, becausethe efflux is completely blocked by human Fc fragments (FIG. 40). Theseobservations indicate the rat BBB FcR recognizes human Fe, in the formeither of human Fc fragments, or the human sequence comprising theCH2-CH3-region of the fusion antibody, which is depicted in FIG. 25. Therodent FcR is known to bind with high affinity to human IgG. See, e.g.,Ober, R. J., Radu, C. G., Ghetie, V. and Ward, E. S. (2001): Differencesin promiscuity for antibody-FcRn interactions across species:implications for therapeutic antibodies. Int Immunol, 13: 1551-1559,incorporated herein by reference.

The in vivo brain efflux in rat shown in FIG. 40 indicates the fusionantibody is able to efflux across the BBB in the brain to blooddirection via the BBB Fc receptor. Therefore, the fusion antibody isshown to perform step 3 in the scheme outlined in FIG. 27. The fusionantibody is also able to perform step 2 of the scheme in FIG. 27, asillustrated by the following examples.

Example 14 Disaggregation of Aβ Plaque by Anti-Aβ ScFv/Chimeric HIRMAbFusion Antibody

Aβ plaque was formed by incubating the Aβ¹⁻⁴⁰ peptide in an orbitalshaker at 37 C for 6 days, and the plaque was collected bycentrifugation. An antibody against the carboxyl terminus (CT) of theAβ¹⁻⁴⁰ peptide was plated in 96-well dishes, as outlined if FIG. 41A. Inparallel, the Aβ plaque was incubated for either 1 or 4 hours at 37 Cwith either the fusion antibody, human IgG1 (hIgG1), or phosphatebuffered saline (PBS), as shown in FIG. 41B, or with the mouse anti-AβMAb, mouse IgG (mIgG), or PBS, as shown in FIG. 41C. The Aβaggregate/fusion antibody, or Aβ aggregate/mouse anti-Aβ MAb complex wasthen added in increasing doses (10, 30, 100 uL, which is equivalent to100, 300, 1000 ng/mL) to the immobilized anti-CT antibody, as outlinedin FIG. 41A. The anti-Aβ ScFv part of the fusion antibody, or the mouseanti-Aβ MAb, binds an epitope on the Aβ¹⁻⁴⁰ peptide near the aminoterminus (NT). Therefore, if plaque is present, then a complex will formbetween anti-CT antibody, the plaque, the fusion antibody, and asecondary antibody coupled to peroxidase for detection of anti-Aβantibody binding to plaque by ELISA. The secondary antibodies used forthe studies in FIGS. 41B and 41C were anti-human and anti-mouse IgG,respectively. The study in FIG. 41 shows (a) that the fusion antibodyselectively binds to Aβ plaque, (b) that a 4 hour incubation of Aβplaque with the fusion antibody nearly completely disaggregates the Aβplaque in a dose-dependent process, and (c) that the anti-Aβ plaquedisaggregation properties of the fusion antibody are as high or higherthan the anti-Aβ plaque disaggregation properties of the original murineanti-Aβ MAb.

The disaggregation of Aβ amyloid plaque shown in FIG. 41, and the Aβbinding data shown in FIGS. 29, 30, and 34, indicates the fusionantibody is able to bind the Aβ plaque of AD, and to disaggregate thisplaque. Therefore, the fusion antibody is shown to perform step 2 in thescheme outlined in FIG. 27.

Example 15 Anti-Aβ ScFv/Chimeric HIRMAb Fusion Protein Binds to AmyloidPlaque in Alzheimers Disease

The fusion antibody was radiolabeled with 125-iodine and chloramine Tand the [¹²⁵I]-fusion protein was applied to microtome sections ofautopsy Alzheimer's disease (AD) brain for 2 hours. The slides werewashed and coated in a darkroom with emulsion. After 1-2 weeks ofexposure in the dark the slides were developed, fixed, washed, andphotographed under bright field with a light microscope (FIG. 42A). Inparallel, sections of the AD brain were immunostained with the hybridomagenerated murine anti-Aβ MAb using peroxidase immunocytochemistry (FIG.42B). The parallel immunocytochemistry and light microscopy of theemulsion autoradiography shows binding of the fusion proteinradiopharmaceutical to the vascular amyloid plaque of AD, and thisbinding is comparable to that observed with immunocytochemistry and themurine antibody against Aβ (FIG. 42).

The fusion protein could be used as an antibody radiopharmaceutical forimaging the amyloid in brain of people suspected of having AD or peoplesuspected of depositing in brain the Aβ amyloid of AD. The fusionantibody could be labeled with a positron emitter for brain scanningusing positron emission tomography (PET), or could be labeled with aradionuclide that could be detected with single photon emission computedtomography (SPECT). For SPECT scanning, the fusion protein can beradiolabeled with 111-indium following conjugation to the fusionantibody of a suitable chelating agent. One such chelating agent is1,4,7,10-tetraazacyclododecane-N,—N′,N″,N′″-tetraacetic acid (DOTA). TheHIRMAb was conjugated with DOTA. The DOTA was obtained from the ParishChemical Company (Oren, Utah), and 16.2 mg of DOTA was dissolved in 0.81ml of water, and 80 μl of 1 M NaOH was added so that the pH is 5.45.This pH has been shown to add approximately 2-10 DOTA chelatormolecules/monoclonal antibody. This solution is cooled to 4° C., and 240μl is removed (4.4 mg) and added to 2.33 mg of sulfo-NHS, whereNHS=N-hydroxysuccinimide, which is obtained from Pierce ChemicalCompany. Then, 8 μl (0.21 mg) ofN-methyl-N′-3-(dimethylaminopropyl)carbodiimide hydrochloride (EDC fromSigma) is added and stirred at 4° C. The pH is adjusted to 7.3 with 0.2M Na₂HPO₄ (pH=9.2). The NHS-DOTA is then added to 8 mg of monoclonalantibody and incubated overnight at room temperature followed bypurification of the DOTA conjugated antibody by gel filtration. Theaffinity of the HIRMAb for the HIR was measured with the ELISA asdescribed in Example 10. The affinity of the DOTA conjugated antibodyfor the HIR is not significantly different from the unconjugatedantibody, as shown in FIG. 43. DOTA-conjugated fusion antibodies can beprepared for radio-labeling with 111-indium and imaging of the targetantigen in brain using standard external detection radio-imagingmethods.

Example 16 Method of Manufacturing IgG Fusion Proteins

The transfection of a eukaryotic cell line with immunoglobulin G (IgG)genes generally involves the co-transfection of the cell line withseparate plasmids encoding the heavy chain (HC) and the light chain (LC)comprising the IgG. In the case of an IgG fusion protein, the geneencoding the recombinant therapeutic protein may be fused to either theHC or LC gene. However, this co-transfection approach makes it difficultto select a cell line that has equally high integration of both the HCand LC-fusion genes, or the HC-fusion and LC genes. The preferredapproach to manufacturing the fusion protein is the production of a cellline that is permanently transfected with a single plasmid DNA thatcontains all the required genes on a single strand of DNA, including theHC-fusion protein gene, the LC gene, the selection gene, e.g. neo, andthe amplification gene, e.g. the dihydrofolate reductase gene. As shownin the diagram of the fusion protein tandem vector in FIG. 24, theHC-fusion gene, the LC gene, the neo gene, and the DHFR gene are allunder the control of separate, but tandem promoters and separate buttandem transcription termination sequences. Therefore, all genes areequally integrated into the host cell genome, including the fusion geneof the therapeutic protein and either the HC or LC IgG gene.

Example 17 Treatment of Parkinsons Disease with a Fusion Antibody thatCrosses the BBB

The neurodegeneration of Parkinson's disease (PD) is caused by thegradual accumulation of protein aggregates called Lewy bodies, which arederived from α-synuclein and parkin proteins. Accordingly, activeimmunization of patients with PD against proteins such as α-synuclein,or parkin, has been proposed. Active immunization of PD may likelyencounter the same difficulties as in the active immunization of AD. Ifthe BBB is not disrupted, then the anti-α-synuclein, or anti-parkin,antibodies in the blood that are generated with the immunization programwill not cross the BBB, and not be able to access the protein aggregatesin brain. Or, if the adjuvant administered in the active immunizationprogram causes disruption of the BBB, then toxic side effects will begenerated. A panel of monoclonal antibodies against α-synuclein orparkin can be generated, and the antibody that disaggregates Lewy bodiescan be selected for production of a ScFv. A fusion antibody of thechimeric HIRMAb and the anti-α-synuclein ScFv can be produced fortreatment of PD.

Example 18 Treatment of Huntington's Disease with a Fusion Antibody thatCrosses the BBB

The neurodegeneration of Huntington's disease (HD) is caused by thegradual accumulation of protein aggregates, which are derived from thehuntingtin protein. Active immunization of patients with HD against thehuntingtin protein has been proposed. Active immunization of HD againstthe huntingtin protein may likely encounter the same difficulties as inthe active immunization of AD. If the BBB is not disrupted, then theanti-huntingtin antibodies in the blood that are generated with theimmunization program will not cross the BBB, and not be able to accessthe huntingtin aggregates in brain. Or, if the adjuvant administered inthe active immunization program causes disruption of the BBB, then toxicside effects will be generated. A panel of monoclonal antibodies againstthe huntingtin protein can be generated, and the antibody thatdisaggregates huntingtin aggregates can be selected for production of anScFv. A fusion antibody of the chimeric HIRMAb and the anti-huntingtinScFv can be produced for treatment of HD.

Example 19 Treatment of Mad Cow Disease with a Fusion Antibody thatCrosses the BBB

The neurodegeneration of mad cow disease is caused by the gradualaccumulation of protein aggregates, which are derived from the prionprotein (Prp). Active immunization of patients with mad cow diseaseagainst the prp protein has been proposed. Active immunization ofpatients with mad cow disease against the Prp protein may likelyencounter the same difficulties as in the active immunization of AD. Ifthe BBB is not disrupted, then the anti-prp antibodies in the blood thatare generated with the immunization program will not cross the BBB, andnot be able to access the prp aggregates in brain. Or, if the adjuvantadministered in the active immunization program causes disruption of theBBB, then toxic side effects will be generated. A panel of monoclonalantibodies against the prp protein can be generated, and the antibodythat disaggregates pip amyloid aggregates can be selected for productionof an ScFv. A fusion antibody of the chimeric HIRMAb and the anti-prpScFv can be produced for treatment of mad cow disease.

Example 20 Treatment of West Nile Encephalitis with a Fusion Antibodythat Crosses the BBB

The West nile virus infects the brain and causes severe encephalitis.Antibodies directed against the envelope protein of the virus blockviral replication. See, e.g., Chung, K. M., et al. (2006): “Antibodiesagainst West Nile Virus (WNV) nonstructural protein NS1 prevent lethalinfection through Fc gamma receptor-dependent and -independentmechanisms,” J Virol, 80: 1340-1351, incorporated herein by reference.However, such antibodies could not be used to treat the encephalitis ofWest nile virus infection, because the antibodies do not cross the BBB,as depicted in FIG. 44. Monoclonal antibodies against the West nilevirus envelope (E) protein are particularly effective in neutralizingWNV infection of cells. A fusion antibody of the chimeric HIRMAb and theanti-envelope antibody can be produced for treatment of West Nile virusencephalitis. As depicted in FIG. 44, the fusion antibody first bindsthe BBB insulin receptor to trigger transport into brain, where theanti-WNV antibody part of the hybrid molecule then neutralizes the WNVin brain behind the BBB.

The synthetic gene encoding the VH of the E16 MAb against the E proteinof the WNV was produced by PCR (FIG. 45A). The VH gene was constructedfrom a series of 8 oligodeoxynucleotides (ODNs), which were designedbased on the sequence of the E16 MAb VH (Genbank DQ083997) and customordered, and the sequences of the ODNs producing the VH are given inTable 4, and in SEQ ID NOS 33-40. The sequences were designed so thatthere are alternating forward and reverse ODNs that cross-hybridize atthe 5′- and 3′-termini of each ODN. The third letter codon issubstituted to reduce the Tm of stable hairpin loops when needed. Theoverlapping ODNs are 75-88 nucleotides (nt) with 24 nt overlap at eachend. The synthetic VH gene is designed to include the appropriaterestriction endonuclease (RE) sites, for subcloning of the VH into asingle chain Fv (ScFv) expression vector, designated pCD-pScFv in FIG.46. The VH of the E16 MAb was cloned by PCR as demonstrated by theethidium bromide stain of an agarose gel following electrophoresis (FIG.45A).

The PCR-generated anti-WNV VH cDNA (FIG. 45A) was subcloned intopCR-Script, maxi-prepped, and subjected to DNA sequencing in bothdirections with T7 and T3 sequencing primers. The results showed the VHgene was successfully cloned with the nucleotide sequence given in SEQID NO: 41. The predicted amino acid sequence of the anti-WNV VH is givenin SEQ ID NO: 42 and in FIG. 47.

In parallel, the gene encoding the VL of the E16 anti-WNV MAb wasproduced by PCR (FIG. 45B). The VL gene was constructed from a series of8 ODNs, which were designed based on the sequence of the E16 MAb VL(Genbank DQ083998). The sequences of the custom ordered synthetic ODNsare given in Table 5, and in SEQ ID NOS 43-50. The synthetic VL gene isdesigned to include REs (HindIII, NotI) at the 5′- and 3′-termini forsubcloning into the pCD-ScFv expression vector as shown in FIG. 46. TheVL of the E16 MAb was cloned by PCR as demonstrated by the ethidiumbromide stain of an agarose gel following electrophoresis (FIG. 45B).

The PCR-generated anti-WNV VL cDNA (FIG. 45B) was subcloned intopCR-Script, maxi-prepped, and subjected to DNA sequencing in bothdirections with T7 and T3 sequencing primers. The results showed the VLgene was successfully cloned with the nucleotide sequence given in SEQID NO: 51. The predicted amino acid sequence of the anti-WNV VL is givenin SEQ ID NO: 52.

Having engineered the genes encoding the VH and VL of the anti-WNVantibody, it was then possible to engineer a new cDNA encoding a ScFvantibody, whereby the VH and VL formed a single polypeptide via a commonpeptide linker. To enable expression of the anti-WNV ScFv in host cells,an expression plasmid was engineered, designated pCD-ScFv. The pCD-ScFvvector is opened with AfeI and HindIII to release the non-related VHgene (FIG. 46). The VH generated by PCR (FIG. 45A) is digested withHindIII, and then ligated into the pCD-ScFv with T4 ligase, in framewith the eukaryotic signal peptide and the 17 amino acid linker joiningthe VH and VL in the pCD-ScFv vector (FIG. 46). The new pCD-ScFv, andthe PCR generated VL, are digested with HindIII and NotI, and the VL isinserted into the vector with T4 ligase to produce pCD-WNV-ScFv, theanti-WNV ScFv expression vector (FIG. 46). Subcloning in the pCD-ScFvplasmid places the c-myc epitope of the 9E10 MAb at the carboxylterminus of the ScFv; this epitope is comprised of the following10-amino acid sequence: EQKLISEEDL (SEQ ID NO: 66). The presence of thissequence and the availability of the 9E10 anti-c-myc mouse MAb allowsfor detection of the anti-WNV ScFv by Western blotting. The pCD-ScFvexpression plasmid also encodes a 6-histidine tag following the c-mycepitope, and the (His)6 (SEQ ID NO: 68) allows for purification byimmobilized metal affinity chromatography following expression in COScells. The nucleotide and amino acid sequence of the anti-WNV ScFV aregiven in SEQ ID NO: 53 and SEQ ID NO: 54, respectively (see FIG. 47 foramino acid sequence).

In order to assess the biological activity of the anti-WNV antibody, itwas necessary to produce the portion of the E protein of the WNV thatcontains the epitope of the anti-WNV antibody. This epitope is containedwithin the DIII region between amino acids 296-401 of the viral Eprotein (Genbank NC001563). The DIII gene is produced by PCR, and wasconstructed from a series of 8 ODNs, which were designed and customordered, and the sequences of the ODNs producing the DIII gene are givenin Table 6, and in SEQ ID NOS 55-62. The sequences were designed so thatthere are alternating forward and reverse ODNs that cross-hybridize atthe 5′- and 3′-termini of each ODN. The PCR-generated DIII (FIG. 45C)was subcloned into pCR-Script, maxi-prepped, and subjected to DNAsequencing in both directions with T7 and T3 sequencing primers. Theresults showed the DIII gene was successfully cloned with the nucleotidesequence is given in SEQ ID NO: 63. The predicted amino acid sequence ofthe DIII protein is given in SEQ ID NO: 64.

The synthetic DIII gene is designed to include the appropriaterestriction endonuclease (RE) sites, for subcloning of the DIII into aeukaryotic expression vector, designated pCD-DIII. For engineering ofpCD-DIII, the pCD-ScFv (FIG. 46) is digested with NotI and EcoRI and gelpurified to release the c-myc encoding sequence. In parallel, anartificial linker is produced with the following sequence:5′-GCGGCCGCTGGATCCCATCATCACCATCATCAT TAAGAATTC-3′ (SEQ ID NO: 67), andthis linker is treated with NotI and EcoRI, and ligated into the openedpCD-ScFv to produce an intermediate plasmid, designated pCD-ScFvII. Thelatter is gel purified and digested with AfeI and NotI, and theNotI-digested DIII PCR product (FIG. 45C) is ligated into theintermediate plasmid to generate pCD-DIII.

The WNV ScFv cDNA, generated by PCR with the pCD-ScFv as template, andusing primers that amplify only the ScFv and not the signal peptide, issubcloned into pCD-UHC to produce pCD-HC-BSA, as outlined in FIG. 46.The pCD-UHC encodes the heavy chain (HC) of the chimeric HIRMAb, underthe influence of a human IgG signal peptide, and is linearized with HpaI(FIG. 46). This site is localized at the immediate 3′-end of the HC openreading frame, and enables fusion of the amino terminus of the anti-WNVScFv to the carboxyl terminus of the CH3 domain of the human IgG1constant region of the HIRMAb. The final heavy chain fusion protein isexpressed by the pCD-HC-BSA vector shown in FIG. 46, which will encodethe protein shown in FIG. 47. The amino acid sequence of the HC anti-WNVfusion heavy chain is given in SEQ ID NO: 65. The fusion heavy chain iscomprised of the following domains, as shown in FIG. 47:

-   -   19 amino acid human IgG signal peptide    -   VH of the chimeric HIRMAb    -   Human IgG1 C-region comprised of the CH1, hinge, CH2, and CH3        regions    -   Ser-Ser linker    -   VH of the WNV MAb    -   17 amino acid linker    -   VL of the WNV MAb

The intact fusion antibody that both crosses the BBB and neutralizes theWNV is a hetero-tetrameric molecule comprised of 2 heavy chains, shownin FIG. 47, and 2 light chains, similar to the structure shown in FIG.26. For permanent transfection of a eukaryotic host cell, for themanufacturing of the fusion antibody, Chinese hamster ovary (CHO) DG44cells may be permanently transfected with separate expression plasmidsencoding the fusion heavy chain (HC) and the HIRMAb light chain (LC). Inaddition, it is necessary to transfect the CHO cells with thedihydrofolate reductase (DHFR) gene to allow for isolation of highproducing cell lines via amplification with methotrexate (MTX). In orderto isolate a high producing CHO line, that has commercial value, andcould meet market demand for the WNV BSA, it is necessary to include all3 genes (HC, LC, DHFR) on a single piece of DNA, called a tandem vector(TV). The TV expressing the WNV fusion antibody is called TV-BSA, and isshown in FIG. 48. The TV-BSA is engineered from 3 precursor expressionplasmids: pCD-HC, which is the HC fusion gene outlined in FIG. 46,pCD-LC, which is the HIRMAb LC expression plasmid, and pwtDHFR, whichencodes the wild type (wt) murine DHFR. The TV-BSA will also encode theneomycin resistance gene (neo) for initial selection of transfected CHOlines with G418. The expression cassettes of the 3 genes include thefollowing:

-   -   The HC cassette is comprised of the cytomegalovirus (CMV)        promoter, followed by the HC fusion gene (which includes the        ScFv fused to the 3′-end of the HIRMAb HC), followed by the        bovine growth hormone (BGH) polyA termination sequence    -   The LC cassette is comprised of the CMV promoter, followed by        the LC gene, followed by the BGH polyA termination sequence    -   The DHFR cassette is comprised of the simian virus (SV)40        promoter, followed by the murine DHFR, followed by the hepatitis        B virus (HBV) polyA termination sequence.

The starting point of the genetic engineering of TV-BSA is thepCD-HC-BSA, as outlined in FIG. 48. Site directed mutagenesis (SDM) ofpCD-HC-BSA is performed to introduce an AfeI site at the end of the BGHpolyA+ site of clone pCD-HC-BSA using the Stratagene QuickChange kitwith ODN primers as per the manufacturer's instructions. Theintermediate plasmid named pCD-HC-BSA-AfeI is digested with NruI andtreated with alkaline phosphatase to prevent reclosing. The LCexpression cassette is released from the pCD-LC vector by doubledigestion with NruI and AfeI and gel-purified. The LC cassette issubcloned into the pCD-HC-WNV-AfeI at the NruI site to form thepCD-HC-LC intermediate plasmid (FIG. 48). The DHFR expression cassette,under the influence of the SV40 promoter, is isolated from the pwtDHFRplasmid by SmaI-SalI digestion and gel-purified with Qiagen gelextraction kit. The SalI end is filled with T4 DNA polymerase. ThewtDHFR expression cassette is ligated in the pCD-HC-LC at the AfeI siteto form the TV-BSA (FIG. 48). The identification of both AfeI-SDM andintermediate positive clones, as well as confirmation of theirorientation, is done by restriction endonuclease mapping and DNAsequencing.

Following the genetic engineering of the TV-BSA, and validation by DNAsequencing, CHO cells may be permanently transfected by electroporation.Following amplification with MTX, and dilutional cloning, a highproducing cell line may be isolated, and propagated in a bioreactor formass manufacturing of the fusion antibody against the WNV. This fusionantibody would be the first monoclonal antibody therapeutic engineeredto cross the BBB and neutralize the WNV. Without the ability to crossthe BBB, an anti-WNV MAb cannot access the WNV behind the BBB. Withinthe sanctuary of the brain, provided by the BBB, the WNV may replicatewithin the brain until encephalitis produces severe morbidity andmortality.

TABLE 4 Oligodeoxynucleotides (ODNs) for production of the  anti-WNV VH1) FWD-1 (75-mer) SEQ ID NO: 33CAGGTaCAGCTGCAGCAGTCTGGATCTGAGCTGATGAAGCCTGGGGCCTCAGTaCAGATATCCTGCAAGGCTACT 2) REV-1 (87-mer) SEQ ID NO: 34CTCAAGGCCATGTCCAGGtCTCTGCTTTACCCACTCAATCCAGTAGTCACTGAATGTGTAGCCAGTAGCCTTGCAGGATATCTGtAC 3) FWD-2 (87-mer) SEQ ID NO: 35CAGAGaCCTGGACATGGCCTTGAGTGGATTGGAGATATTTTATGTGGAACTGGTAGAACTAGATACAATGAGAAGTTAAAaGCaATG 4) REV-2 (88-mer) SEQ ID NO: 36CAGATGTCAGGCTGCTtAGTTGCATGAAGGCTGTGTTGGAGGATGTATCTGCAGTGAATGTGGCCATtGCtTTTAACTTCTCATTGTA 5) FWD-3 (87-mer) SEQ ID NO: 37TGCAACTaAGCAGCCTGACATCTGAGGACTCaGCaGTCTATTACTGTGCAAGATCGGCaTCATATGGTGATTACGCTGACTACTGGG 6) REV-3 (77-mer) SEQ ID NO: 38TTCAAGCTTGGGTGTCGTTTTGGCTGAGGAGACTGTGAGAGTGGTGCCATGGCCCCAGTAGTCAGCGTAATCACCAT 7) 5′-PCR FWD (24-mer) SEQ ID NO: 395′-phosphate-CAGGTaCAGCTGCAGCAGTCTGGA 8) 3′-PCR-REV (24-mer)SEQ ID NO: 40 TTCAAGCTTGGGTGTCGTTTTGGC Lower case indicates the 3rdletter codon nucleotide substituted to reduce the Tm of stable hairpinloops to 30-46° C. Overlapping ODNs are 76-88 nt long with 24 nt overlapat both ends. All ODNs were reconfirmed by reverse testing, wherein thecDNA was constructed from the ODNs, and the former translated. Thetranslated protein was identical to the expected one.

Lower case indicates the 3rd letter codon nucleotide substituted toreduce the Tm of stable hairpin loops to 30-46° C. Overlapping ODNs are76-88 nt long with 24 nt overlap at both ends. All ODNs were reconfirmedby reverse testing, wherein the cDNA was constructed from the ODNs, andthe former translated. The translated protein was identical to theexpected one.

TABLE 5 Oligodeoxynucleotides (ODNs) for production of  the anti-WNV VL1) 165-LV-FWD-1 (76-mer) SEQ ID NO: 43TACAAGCTTGAAGAAGGTGAATTTTCAGAAGCACGCGTAGATATCGACATaGTGATGACCCAGTCTCACAAATTCA 2) 165-LV-REV-1 (88-mer) SEQ ID NO: 44GCAGTACTCACATCCTGACTGGCCTTGCAtGTtATGCTGACtCTGTCTCCTACTGATGTGGACATGAATTTGTGAGACTGGGTCATCA 3) 165-LV-FWD-2 (88-mer)SEQ ID NO: 45 GGCCAGTCAGGATGTGAGTACTGCTGTAGCaTGGTATCAACAAAAACCtGGGCAATCTCCTAAACTACTCATTTCCTGGGCATCCACa 4) 165-LV-REV-2 (88-mer)SEQ ID NO: 46 TGGTGAGAGTATAATCTGTCCCAGATCCACTGCCaGTGAAGCGATCgGGtACTCCtGTGTGCCGtGTGGATGCCCAGGAAATGAGTAG 5) 165-LV-FWD-3 (88-mer)SEQ ID NO: 47 CTGGGACAGATTATACTCTCACCATCAGtAGTGTGCAGGCTGAAGACCTaGCACTTTATTACTGTCAGCAACATTATACCACTCCCCT 6) 165-LV-REV-3 (78-mer)SEQ ID NO: 48 GATGCGGCCGCAGCgTCAGCTTTCAGCTCCAGtTTGGTtCCAGCACCGAACGTGAGGGGAGTGGTATAATGTTGCTGA 7) 5′-PCR 165-VL-F SEQ ID NO: 49TACAAGCTTGAAGAAGGTGAATTTTC 8) 3′-PCR 165-VL-R SEQ ID NO: 50GATGCGGCCGCAGCgTCAGCTTTC Lower case indicates the 3rd letter codonnucleotide substituted to reduce the Tm of stable hairpin loops to30-46° C. Overlapping ODNs are 76-88 nt long with 24 nt overlap at bothends. All ODNs were reconfirmed by reverse testing, wherein the cDNA wasconstructed from the ODNs, and the former translated. The translatedprotein was identical to the expected one.

Lower case indicates the 3rd letter codon nucleotide substituted toreduce the Tm of stable hairpin loops to 30-46° C. Overlapping ODNs are76-88 nt long with 24 nt overlap at both ends. All ODNs were reconfirmedby reverse testing, wherein the cDNA was constructed from the ODNs, andthe former translated. The translated protein was identical to theexpected one.

TABLE 6 Oliogodeoxynucleotides (ODNs) for production of DIII of WNV E protein 1) DIII-FWD-1 (68-MER) SEQ ID NO: 55CAGCTGAAGGGAACAACATATGGAGTCTGCTCAAAAGCTTT CAAATTCGCTAGGACTCCCGCTGACAC2) DIII-REV-1 (80-MER) SEQ ID NO: 56TGCAGGGACCGTCTGTTCCAGTATATTGCAGTTCCAACACCACAGTTCCATGTCCAGTGTCAGCGGGAGTCCTAGCGAAT 3) DIII-FWD-2 (80-MER)SEQ ID NO: 57 ATACTGGAACAGACGGTCCCTGCAAAGTGCCCATTTCTTCCGTAGCTTCCCTGAATGACCTCACACCTGTTGGAAGACTG 4) DIII-REV-2 (80-MER)SEQ ID NO: 58 TCAATCAAAACCTTCGAGTTGGCTGTGGCTACAGACACAAATGGATTCACGGTCACCAGTCTTCCAACAGGTGTGAGGTC 5) DIII-FWD-3 (80-MER)SEQ ID NO: 59 AGCCAACTCGAAGGTTTTGATTGAACTCGAACCCCCGTTTGGTGACTCTTACATCGTGGTGGGAAGAGGAGAACAGCAGA 6) DIII-REV-3 (70-MER)SEQ ID NO: 60 GTAGCGGCCGCAGCATCAGCTCCAGATTTGTGCCAGTGATGGTTTATCTGCTGTTCTCCTCTTCCCACCA 7) 5′-PCR DIII-FWD (25-MER) SEQ ID NO: 61CAGCTGAAGGGAACAACATATGGAG 8) 5′-PCR DLII-REV (24-MER) SEQ ID NO: 62GTAGCGGCCGCAGCATCAGCTCCAG

Example 21 Treatment of Neuro-AIDS with an Antibody that Crosses the BBB

The human immunodeficiency virus (HIV) causes acquired immune deficiencysyndrome (AIDS), and the HIV infects the brain to cause dementia andother symptoms of neuro-AIDS. The tumor necrosis factor (TNF)-relatedapoptosis-inducing ligand (TRAIL) induces neuronal death in neuro-AIDS,and administration of an anti-TRAIL antibody blocks the neuronalapoptosis in the HIV-infected brain following systemic administration oflipopolysaccharide, which causes BBB disruption. However, disrupting theBBB is not a safe method for administration of antibodies to the brain,because serum proteins are toxic to the brain. A fusion antibody of thechimeric HIRMAb and the anti-TRAIL ScFv antibody can be produced fortreatment of neuro-AIDS. The treatment of AIDS infection of the braincould also use an anti-carbohydrate, or anti-glycan antibody. Monoclonalantibodies against the carbohydrate portion of Schistosoma mansoni arepotent inhibitors of HIV proliferation. A fusion antibody of thechimeric HIRMAb and the anti-glycan antibody can be produced for thetreatment of viral infection of the brain, including HIV-1 infection ofthe brain.

Example 22 Treatment of Brain or Spinal Cord Injury or Stroke withFusion Antibody that Crosses the BBB

The recovery of the damaged brain following brain injury, spinal cordinjury, or stroke is inhibited by a naturally occurring protein in thebrain, nogo-A. Inhibition of nogo-A by a monoclonal antibody increasesthe functional recovery following brain damage. See, e.g., Buchli, A. D.and Schwab, M. E. (2005): Inhibition of Nogo: a key strategy to increaseregeneration, plasticity and functional recovery of the lesioned centralnervous system. Ann Med, 37: 556-567, incorporated herein by reference.The BBB is disrupted as the brain heals following injury, andsystemically administered anti-nogo-A antibodies might penetrate thebrain during this time window. However, the BBB closes following theperiod of BBB disruption, and during this time it is still necessary toinhibit endogenous nogo-A in the brain. A fusion antibody of thechimeric HIRMAb and an anti-nogoA antibody can be produced for treatmentof the recovery period from brain injury, spinal cord injury, or stroke.

Example 23 Treatment of Cancer Metastatic to the Brain with a FusionAntibody that Crosses the BBB

The growth of HER2-positive breast cancer cells is inhibited by amonoclonal antibody against HER2. Breast cancer often metastasizes tothe brain, where the breast cancer cells reside behind the BBB. In thissetting, the anti-HER2 antibodies are not able to inhibit growth of thebreast cancer in the brain, because the antibodies do not cross the BBB.See, e.g., Duchnowska, R. and Szczylik, C. (2005): Central nervoussystem metastases in breast cancer patients administered trastuzumab.Cancer Treat Rev, 31: 312-318, incorporated herein by reference. Afusion antibody of the chimeric HIRMAb and an anti-HER2 antibody can beproduced for treatment of metastatic breast cancer of the brain. Otherepithelial cancers, such as small cell lung cancer (SCLC) expresstumor-associated antigens (TAA) that are glycolipids or gangliosides.Monoclonal antibodies against the TAA glycolipids induce apoptosis andsuppress cell growth of SCLC. A fusion antibody of the chimeric HIRMAband an anti-glycolipid antibody can be produced for treatment of themetastatic cancer of the brain.

Example 24 Treatment of Brain Cancer with a Fusion Antibody that Crossesthe BBB

The growth of brain cancer cells is stimulated by certain trophicfactors such as epidermal growth factor (EGF) or hepatocyte growthfactor (HGF). Inhibition of either EGF or HGF is a treatment strategyfor brain cancer, and these growth factors are inhibited by growthfactor-specific monoclonal antibodies. However, the antibodies do notcross the BBB, which is intact in brain cancer. Consequently, thesystemic administration of an anti-trophic factor antibody does notsuppress growth of intra-cranial brain cancer. See, e.g., Sampson, J.H., et al. (2000): Unarmed, tumor-specific monoclonal antibodyeffectively treats brain tumors. Proc Natl Acad Sci USA, 97: 7503-7508,incorporated herein by reference. A fusion antibody of the chimericHIRMAb and an anti-growth factor antibody can be produced for treatmentof brain cancer.

Example 25 Treatment of Multiple Sclerosis with a Fusion Antibody thatCrosses the BBB

Multiple sclerosis (MS) is associated with the loss of myelin in thebrain. Therapy of MS aims to increase remyelination, which can bepromoted by monoclonal antibodies directed against oligodendrocytesurface antigens. See, e.g., Warrington, A. E., et al. (2000): Humanmonoclonal antibodies reactive to oligodendrocytes promote remyelinationin a model of multiple sclerosis. Proc Natl Acad Sci USA, 97: 6820-6825,incorporated herein by reference. Although the BBB may become disruptedin MS, this disruption is intermittent. A fusion antibody of thechimeric HIRMAb and an anti-human oligodendrocyte surface antigenantibody can be produced for treatment of MS.

Example 26 Treatment of Brain Disease with a Fusion Antibody thatCrosses the BBB Preferentially in the Blood to Brain Direction

In the treatment of brain aggregate disease, such as Alzheimers disease,Parkinsons disease, Huntingtons disease, or mad cow disease, the intentis to clear from the brain the aggregated protein, via efflux across theBBB via the BBB FcR (FIG. 27). However, in the case of other braindiseases, such as taught in Examples 20-25, the intent is to prolong theresidence time of the antibody therapeutic in brain. In this case, theefflux of the fusion antibody from brain can be minimized by eliminatingthe part of the fusion antibody that is the binding site of the BBB FcRefflux system, which is found in the regions encompassing CH1, CH2, andCH3 shown in FIGS. 25 and 26. In this case, the genes encoding the heavychain of the fusion antibody can be engineered so as to eliminate any orall parts of the CH1, CH2, or CH3 regions of the heavy chain. The ScFvprotein can be fused to the carboxyl terminus of the CH3 region, asoutlined in FIGS. 25-26, or the ScFv protein can be fused to thecarboxyl terminus of the hinge, CH1, or CH2 regions of the heavy chain.

Example 27 Diagnosis of Alzheimers Disease with a Blood Test Based onMeasurement of Fusion Antibody-Aβ Complexes

The administration of the fusion antibody to patients with AD isexpected to lead to the formation in blood of complexes between thefusion antibody and the Aβ peptide, following efflux of the complex frombrain to blood, as depicted in FIG. 27. Such complexes should form ingreater quantities in the patient with amyloid build-up in the brain,which is specific for either AD, or pre-AD conditions, such as mildcognitive impairment. The amyloid in brain that causes AD is known toaccumulate for years before the onset of symptoms. Therefore, theformation of increased complexes between the fusion antibody and Aβ,following the administration of the fusion antibody could lead not onlyto the detection of active AD, but also to the detection of individualsat risk for the later development of AD. The fusion antibody-Aβ complexin human blood could be quantitated with a sandwich based ELISA usingstandard methodology. In one such embodiment of the assay, an anti-Aβantibody could be plated, followed by capture of the fusion antibody-Aβcomplex. Since the fusion antibody binds to the amino terminal portionof Aβ, then the capture antibody would be selected so that there isbinding to the carboxyl terminal portion of Aβ, similar to that used inthe disaggregation assay shown in FIG. 42A, and discussed in Example 14.The second antibody in the sandwich assay could be an anti-human IgGantibody, as illustrated in FIG. 42A, and Example 14.

Example 28 Treatment of Brain Cancer with Fusion AntibodyRadiopharmaceuticals

Brain cancers over-express certain surface antigens as compared tonormal brain, and one approach to the radio-therapy of brain cancer isthe administration of a MAb therapeutic that is conjugated with aradionuclide, such as ²¹¹At-labelled anti-tenascin MAb. See, e.g.,McLendon R. E. et al. (1999): Radiotoxicity of systemically administered²¹¹At-labeled human/mouse chimeric monoclonal antibody: a long-termsurvival study with histologic analysis. Int. J. Radiation OncologyBiol. Phys. 45: 491-499, incorporated herein by reference. To facilitatetransport of the MAb therapeutic across the BBB, aradionuclide-conjugated fusion antibody can be produced, which iscomprised of the chimeric HIRMAb and a MAb directed against a braintumor-associated antigen. The exposure of normal brain to the radiationcan be eliminated by conjugating the fusion antibody with anon-radioactive radionuclide, such as ¹⁰B. Following administration ofthe ¹⁰B-labeled fusion antibody, and efflux of the antibody from normalbrain, the brain tumor may be selectively irradiated with low energythermal neutrons, which generates local alpha ray-irradiation of thebrain cancer. See, e.g., Barth, R. F. et al. (1999): Boron neutroncapture therapy of brain tumors: an emerging therapeutic modality.Neurosurg. 44: 433-451, incorporated herein by reference.

What is claimed is:
 1. A method of diagnosing a brain disordercomprising detecting a signal emitted by a composition in the centralnervous system (CNS) of an individual, wherein the composition comprisesa fusion antibody that: (i) comprises a first antibody and a secondantibody, wherein the first antibody is an ScFv antibody that is linkedto the carboxy terminus of the second antibody; (ii) is capable ofcrossing the blood-brain barrier (BBB) from the blood to the brain bybinding an endogenous receptor of the BBB; (iii) comprises an Fc regionthat enables crossing the BBB from the brain to the blood by binding toan Fc receptor; and (iv) interacts with a pathological substanceassociated with the brain disorder, wherein the ScFv retains at least10% of its affinity compared to the affinity of the tetramericimmunoglobulin from which the ScFv can be derived.
 2. The method ofclaim 1, wherein the pathological substance is a protein.
 3. The methodof claim 2, wherein the protein is Aβ amyloid, α-synuclein, huntingtinprotein, PrP prion protein, West Nile envelope protein, tumor necrosisfactor (TNF) related apoptosis inducing ligand (TRAIL), Nogo A, HER2,epidermal growth factor receptor (EGFR), hepatocyte growth factor (HGF),or oligodendrocyte surface antigen.
 4. The method of claim 3, whereinthe protein is Aβ amyloid.
 5. The method of claim 1, wherein thecomposition is labeled with a substance that emits the signal.
 6. Themethod of claim 5, wherein the substance that emits the signal isselected from the group consisting of positron emitters, radionuclide,and magnetic substances.
 7. The method of claim 1, further comprisingsystemically administering the composition to the individual prior tothe detecting step.
 8. The method of claim 1, wherein the detecting stepcomprises using positron emission tomography (PET), single photonemission computed tomography (SPECT), or magnetic resonance imaging(MRI).
 9. The method of claim 1, wherein the detecting step comprisesquantitation by a sandwich enzyme-linked immunosorbent assay (ELISA).10. The method of claim 1, wherein the pathological substance isselected from the group consisting of a protein, a nucleic acid, acarbohydrate, a carbohydrate polymer, a lipid, a glycolipid, a smallmolecule, and a combination thereof.
 11. The method of claim 1, whereinthe endogenous receptor of the BBB is an insulin receptor, transferrinreceptor, leptin receptor, lipoprotein receptor, or IGF receptor. 12.The method of claim 1, wherein the fusion antibody is able to cross theBBB by binding an insulin receptor.
 13. The method of claim 1, whereinthe ScFv is capable of binding the endogenous receptor of the BBB. 14.The method of claim 1, wherein the ScFv is fused at its amino terminusto the carboxy terminus of the heavy chain of the second antibody. 15.The method of claim 1, wherein the ScFv is fused at its amino terminusto the carboxy terminus of the light chain of the second antibody. 16.The method of claim 1, wherein the ScFv interacts with the pathologicalsubstance associated with the brain disorder.
 17. The method of claim 1,wherein the ScFv is able to bind to amyloid β (Aβ) peptide.
 18. Themethod of claim 1, wherein the ScFv dissociation constant of the ScFvfor its antigen is less than 100 nM.
 19. The method of claim 1, whereinthe second antibody interacts with the pathological substance associatedwith the brain disorder.
 20. The method of claim 1, wherein the secondantibody is able to bind to amyloid β (Aβ) peptide.
 21. The method ofclaim 1, wherein the second antibody is a monoclonal antibody.
 22. Themethod of claim 1, wherein the first antibody is covalently linked tothe carboxy terminus of the second antibody.
 23. The method of claim 1,wherein the brain disorder is Alzheimer's disease, Parkinson's disease,Huntington's disease, bovine spongiform encephalopathy, West Nile virusencephalitis, Neuro-AIDS, brain injury, spinal cord injury, metastaticcancer of the brain, metastatic breast cancer of the brain, primarycancer of the brain, or multiple sclerosis.